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Council for Tobacco Research

Report of the Council for Tobacco Research-U.S.A., Inc. [St]

Date: 1982
Length: 60 pages
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L•:X'1'1(A COPY REPORT Of THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. 1982
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1, Organization and Policy The Council for Tobacco Rasearch-U.S.A., Inc. is tbe sponsoring aycucy of a proFaJn of research into questions of tobacco we and hcalth. It is tbe out- growth of ao ortanir..tioo formed early in 1954 by tobacco manufacturcn, jrowera and warehowemen. Research support has been mainly through a pru- yram of lraota-io-aid supplemented by contracu for rescarch with irutitutionm and laborawrie.. TLe Council does oot operate any researcb facility. The ScicntiCc Advisory Board to The Council meets regularly to cvaluaie applicationa for research aupport, judging tbcro solely on the basis of acicotillc mcrit aod rdevanos. The Council awards research grants to independent scientists who are as- sured complete scieatiflc frocdom in conducting their atudies. Grantces alone are reapoou" tor reportinj or publishint their flodinp in the acccptcd scicn- tibc raaoacr - throuslt modiul aqd acicntitk jouroal. and socictica. WlLLIAM D. Noaas Chairman ~ . ~ 198'l RRI'OR'1' p ~ F ~ o d i ~ C TiIE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. TIIE COUNCIL FOR TOItAC/'O IIESF.AIit'fl-U.S..4., Inc. 110 Eut 59th Street, New York, N.Y. 10022
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c Slal•:NTtI•'ll: ADVISUItY 1tl)AIiI) to Thc Council for Tobacco Rcacarch-U.S.A., Inc. as of December 31, 1992 LEON O. JACOBSON, M.D., Chuirutun lusrph Regcwlcirt Pru/c'ssur uJ lliulugic-ul Scirrn rs ( rinrritus ) I'ruJcssur of rlti• Departmcnt u/ Mrdiccinc (rrncritus) University of Chicago Chicago, Illinois RICf IARD J. BING, M.D. Dirrc•tur u/ fs'.rprrirnrnrul Cardiology ruuf St it•nti/ic /).•vrloq„nc'nl Huntington McJical Research Institutc, PasaJcna, California PruJrssur u/ Medicine (rrneriuo') University of Southern California School of Medicine Los Angcles, California ROSWELL K. BOUTWELL, 1'tt.D. Pru/rasor u/ Onculugy McArdlc Laboratory for Cancer Research Univcrsity of Wisconsin Madison, Wisconsin DRUMMOND 11. LIOWDL•N, M.D. l'rolrssur and Ilrad DcPartmcnt of Pathology Unrversity of Manitoba I Ieahh Sciences Center Winnipeg, Canada MICHAEL J. BRENNAN, M.D. PresiJent and Mulical Director Michigan Cancer Foundation Dctroit, Michigan JOSEPH D. FELDMAN, M.D. Mernber, Research lnstitute of Scripps Clinic Scripps Clinic and Research Foundation La JoUa, California WILLIAM U. GARDNER, Ptt.D. E. K. !lunt Pru/essar u/ .1 natuury ( cnrrritus ) Yale University School of Medicine New tlaven, Connecticut PL'rLR M. I LOWLEY, M.D. l.aboratury uf I'athalogy National Canccr lnstitutc Bethesda, Maryland 11ENRY 1'. I.YNCf f, M.D. l'ru/cssur and Chairman L)cpartment of Preventive Medicine and Public Health Crcightun Univcrsity School of Medicine Umaha, Nebraska G. BARRY I'IERCE, M.D. Aiurrirurt Cunrrr Scx4rty Crurrnniul Rr3rurc h NruJrssur llnivcr.ity of Colorado l{eallh Sclcncc\ Cenlcr 1)cnvcr, CuluraJu GORDON lf. SATO. Ptr.D. f'rujr.r.wr u/ hiulugy University of California, San Diego La Jolla, C'afifurni•a SIIL•I.tX)N C. SUMMIiRS Sric•utifir Uirrrlur, Thc C'uuncil fur Tobacco Rcscarch-U.S.A., Inc. ('Gni4 ul l'ru/rs,fur u/ l'ur/wlugy Collcgc of Physicians & Surgcuns of Colurnbia Univcrsity New York, New York ScieutiGc StrfC of '1'lrc• Council SHELDON C. SOMMERS, M.D. Scic'rui/ic Director ROBERT C. HCKKETT, Pu.D. Research Dirrrtur DONALD II. FORD, Ptt.D. VINCENT F. LISANTI, l).M.D. if ssuuutr Kc•srarrh Director Associutc Kcsc•un b Dirrc rur DAVID STONE, 1'tt.D. Associutr Krarun'/r Dircc lur
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t. CONTENTS Introduction . ... . . . . • • • • • 5 Abstracts of Rcports . • . . . • • • • • 7 Canccr-Rclatcd Studics . . - • • • • • • • • • • 7 The Rcspiratory System . . . . • • • • • • • • 23 Heart and Cireulation . • . . . • • • • • • • • 49 Ncuropharmacology and Physiology . . . . . - . . . 60 Pharmacology and Biochemistry . . . . . • • • . . 65 Immunology and Adaptivc Mcchanisms . . . • • • • 78 Epidcmiology . . . . . . . . . . . . . . . . 89 Activc I'rojccts . . . . . • . • • • • • • • • • • • 95 Cornplctcd Projccts . . . . . • • • . • • . • . . . 105 Index of Principal Investigators . . . . . . . . . • . . IlK Indcx of Scuiur Authors . . . ... . . . • • • • . . . 119 Introduction ® ~ H ~ ~ 'I he ('uuncil tor Tobacco Kescarch's program cxprndcd turthcr tTP1982 in rc.ponx tu a gruwing number of solid and ullcn yultc imgin.ruvc grant apphc:Itiun. 111.11 cuntinucd to comc Iruns indepcndent xnnusks. It xcnted clCar Junog the ycar Ihal evcn with the vasl amount ul rc-scarch done tur ncarty thfcc dccadr. nt/o yucslwns rclatcd to smoking .nd hc.rlth, many in the sci- cntr6c cumnnmuy Inhcvc impurlam gaps in knowlcddc r.mam to be lilkd. 'Ilxrctuic, rcll.cung whal appcars 1o be an in.rc.xd tnlcresl in smoking .md hcahh f.x:uch rmung scwnlhls, the Council suhslanually enlrrgcd its an- nu.t1 cumnuUninl. Ilic acltun :J.o rclkctrd the Councd's suptxrrl lor the pl.ns rnd Jc+ir.N ut ih SaanUtic Adviwty Board, which /s alw:ryN on the luokout lur yuwhh.d mvc.lig.tlurs :u vtutly •pectliC jrYas Ih.A are corlvidered irnpurunl. S-u.r 1'fl 1. the ( uuncJ ha% piuvr.lcd 17b.1MM11MM! La iLs Ics.jrch prugram, wlu.h I. IK•Iw•vcJ lu Iw the I.ug.•.1 .md mua ritlcn.rvc ul its ktnd in the wurld. Ifrlay, a..u Ih: oW.a. Ihr piul;r.urr cmph.f.ucv rrscjfih by independenl sei- culnl. In Ihnl; C.ItJN/v.1~/1IJr Ih.ct.c. arxl chrunic pulnwnary atbucrlK. I bc.r luu.l, wce.• lur M_'9 urnginal granh aoJ nurncruus renewals, smce iuany ol the piul:cl. w.fe Iur 1w.f rnd Ihtre yc:us ..r lun6rr. the ('uuncd h.r-, Ihiuugh the y:..n, Iun.l.•d 466 i..e.u:has in 272 nkJt.:rl schouls, husprlah jnd tr.c.urh m.utuuun.. A nu•.r+uic ot Ihc ('ouiw'd•s vuulnhuUUn Io the jdvan:cmerH uf s.iculilic Inlunn.iUon u.ten m the nun1hcr ul rcpurl% puhh.hed by gnntccs Ihal .c- kouwlcdgc ('uuikil supporl. Ihal ligurc was'_,168 w of 1?rccutbcr 11, I9M_'. the CouncJ r.m:nns Jcflicalcd to cuntinurncc of h% resc.rch ctlort.
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Abstracts of Reports Fulkrwing art ahatracts, approved by the authoo, of rcprxts rnr ncw rescarch ackrrowk;dging sulr{xxt from The Council that have appcucJ in xicnti(ic j.wrnals sincc publication of thc 19g1 Rcport. The name of the grant rccrprcnt is in rtalics. Thc abstracts arc grouped under these headings:t. Canccr-Rclatcd StuJics,11. llrc Rcspiratory Systcm, 111. Heut and Circulation. IV. Ncunrpharmaculugy and Physiulogy. V. Pharmacology and Bwrchcmiury, VI. ImmurKrkrgy and Adaptive Mechanisms, VII. Epidemiology. I. Cancer-Related Studies MECI(ANISM OF ACCION OF fiL•NZO/aIPYR(sNf•. AND NICOTINE ON HORMONE PKODUC"CION BY RAT PITUITARY TUMOR CELLS Akhough hrxTnoncs have bccn associated with induction and pwEres+run of tu- nrxs in many cxpcnmcnul systcros. the role of hurrnones in the process uf initiation and prugrcssiun uf carcinugcncsis is rNx clcarly dchned as yet. In thc present attcmpt to wxkrstand tlr- rncchamsm of action of bcnm(a)pyrcnc (fiaP), a cyclic anunauc hydakarbrm. anJ that of nrcwrnc, the tobacco alkaloid, the clfectu of thesc agents on Ixulactin (1'KI.) arrJ gruwth IKxnrrxrc (Gil) synthcsu by rat pquuary IuuNx cells in culture (GI I cclL ) were audicd. Treatment of Gil cclls with mcrxmc (U.1-XK) µl:/ml) ncuhcr affcctcJ the growth nr)r significantly altcred the gcnrral pattern uf humrunc pu><lucurxt in these cells. BaP at concentrations greater than Sµgnnl xrevcrsrbly inlxhitcd the gruwth uf these cclls. The sublethal cunccntrahuns of HaP. which did not al(cct either (I) cell growth, or (2) amino acid transfxwrt rx (3) aaal protein synthcsrs or Jcgradatirxt, did however inhibit spccihcally hurmunc synthc.is by these cells. More interestingly, axrcentrauons of nicotrrre, which did na affect either cell growth or Ixxnwnc syntlresrs, modulated both of thcse cellular prucc»es in thc prcscnce of HaP. A cunccntratHm dcpcndent sonwlauon uf micrownsal iSaP uwnaraygcnax activity was uhscrved in nicotine or BaP ucatcd cells. The cllccts of ::xsc subsunccs on stimulation uf IIaP mrxwuxygcnase activity scems to be additivc. Nicotine alvr cn- hanccd the asxkiauun uf raJioa.uvity (presumably /'H It3aP nkt,tb.ddcowith DNA m )'H/UaP trcatcJ cells. 1t is concludcd that niatxrnc by uself did skw Jcnwmstratc any cyt.wuxic cfl'cct nur inlluence hurrrwne synthesu in GH cclls. IJuwevcr. nlarNlnc atimulatcJ Bal' munrwaygcnax activity anJ the rrueracuun ul 1'H1BaP nktububtcs wrth ccllular UNA anJ alsu modulatcJ BaP irnlurcJ intubitrun u( MxrrwMk synth.su m Gil cells. CTakrabani, S., Hai>LS- S. D. and Bra»wi, l1. K. !!rw'hemrul und 8rupbi•sirul Rrararrh C'r,m.aunrruuuru IOK(.) S'M-(03, IV%:. Frum t)rc I-abxatury of Ph:unr.rculugy, H:warJ SrM.d of D.nt,rl Medicine anJ Dc - partnknt uf 1'harrnaculugy, FluvarJ MrJrcal SaNrul. Busam. 7
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POSITIVE C'ORRELATION BGTWEEN HIGII ARYL IIYI)R<KAR(iON HYDROXYLASE ACTIVITY AND PRIMARY LUNG CANCER AS ANALY'LF3) IN CRYOPRGSERVED LYMPHOCYTES Blood sampks from SI paticnts at the Veterans Aduunisoratiun 1lu.pital, I luur lon, were collected, cakd, and scm tu Microbiological Asx>tiatcs, Bcthcxla, MD. where the lymphocytes werc isolated and cryoprcxrvcd :n - I'JA°C bclure cxuoina- tion. AI the timc of assay, lymphocyte samples were simultaneously thawed, phytolrc- ongglutinin activated, and analyzed (or benz(a)anthracenc-induccd aryl hydrucarbun hydroxylase (AHH) kvels, ('H/thymidinc incor(wration, and reduced mcuunanridc adenirse dinuckotide-dependent cytuchrome b, (cytuchrume c) rcductasc activity. Uc- lennirrations werc made at both 96 and 12U hr in culture, and pcak activittcs wcrc compared among the SI individuals who cxprc»ed such lesions as syuatnous ccll carcirwmas (22`b), adcnocarcinomas (14%), oat ccll carcinomas (64u), chronic ub- srnactivc pulmonary disease (22'b), and other nonmalignant dueascs. O) the 14 high- est AHH/cytuchromc c activities obscrvcd, all were found in paticnu with pruuary lung cancer. Mean AHH/cytochrome c activities were U. M9 fur lung cancer patients (a Iot:J of 21) and 0.47 for noncancer patients (a lotal of 30). No relationship was obscrvcd belwcen AHH/cytochtomc c activity and age of paticnt, number of cibarcttes srrsuked, family history of cancer, location or histological type of twnur, or Icvcl of phytohernagglutinin blastogcnesis (I'HlthymWine cpm/cytuchrunk c). Although the present communication presents data which show a striking correlation between the prcscnce of pulnwrsary carcinomas and high AHII levels in lytnphucytcs tsulatcd (rum patienu, whether the higher AHH kvcls arc the cause or the result of the primary lung cancer still remains to be determined. Kouri. R.E. rr al. (Micrubiologicrrl Associurrs) Concrr Rrsrwcb 42(12):5030-5037, 1982. O[Aei support: American Cancer Society and the Vctcratu Adnunisltatiun Hu.pttal, 1 iuusuia. From the Dtvisiun of Toxieulogy and Orrculugy, Mtcrubiulugical Asx,ciatcs, Ile- thesda, MD, Departmeru of Biological Sciences, Nonh Tcxas State Univcnity, Den- tun. and the Department of Motlicine, Baylor College of Medtcine, and Veterans Hospiul, Houston. ARYL HYDROCARBON INDUCIBILITY IS NOT ALTERED IN BLADDER CANCER PATIENCS OR THEIR PROGENY The ruk of aryl hydrocarbon hydruxylasc (AHH) urJucrbthty in prcdi.pt.inK perwrrs to cancet has been the subject of considerabk cuntruvcny, with suntc reptms shuwing an increased risk of respiratury cancer in perwns with high AHI I irrducibtltty and others showing no such effect. In the present sttrdy, the pu»ible m0ucrke of AHl I on susceptibility to bladder cancer in humans was carefully investtgatcd. AHH nniuct- bility was mcasured in the cultured lymphcuytes of 16 patients who were tktng lul- luwed a(ter successful treatment for blackkr cancer, in 53 progeny of bladder cancer p.t,cnl., atxl in nutdkd cuntrul.. In both thc progcny and paucnt pupulawm., nu evidence was Irwtkl for a diffcrerxe between the dutnbuuun of AHiI tnduitMltty or induced AHiI activtty ctnnpared to the diuribuhon atnong corurul tndtvtduah. Thus. AHI I aruvity or inducibilhy dtd rwl appear to bc a rnajor dctcrmnant of bladder cancer risk in humans. 1'aigcn, B. rr u(. (Puigrn. A. J lnrrrnuriunul Juurnuf ojCuncrr 23:312-315, 1979. A Urbrr support: National Cancer Institute Frum the Dcpartmcnt of Molecular Biology. Ruswell Park Mcnwnal Institulc. ~ Bulfaks. MONOOXYGENASE AND EPOXIDE HYDROLASE REGULATION IN PRIMARY FtiTAL RAT LIVER CELL CULTURE In this rcpun, various lines of evidence arc presented dcrn,mstratmg Ihat a cyto- chrunx: P450 comparahlc to that of the adult rat liver can be found in /etal hcpauicyte culturc. and that nm appeuancc is strictly controlled by nuticords. The liM.vidcncc cited hcre slwws that the level o( the cyn>Lhromc P450 content in primary Ictal liver cell culturc is rcourkably atablc and does n.N sigmAcantly dtllcr Irutu that hwnJ in Ict.rl hvcr uscd fut.•ulturc prcparattun. Aryl hydnrcarb.m hydruxyl.r.c IA1111) arMl epustdc hydrulasc (El l) Ji uvuUC. arc casrly nx~surabk 10 thc.clls and Icnd tu Jc.rcax sltghtly as a IuwK'ttun of tlK culture duration. High conccntrauutr of phcntbarbual (Ptl l urduces both AI111 and 1i11 acttvitics, whereas hcnt (a)unhraccne t1fAI xts prclcrenn.lly and 2.3,7,1i•tctrachlunxldx ntu-p-dwxin selectively on A)1H acttvtty. Tnns-stiltwne ux- kk atwl ethoxyywn behave as selective induccrs of EH. Other evtdcn.e shuws that AIIFI and EII activities in fetal liver cells uc mudthed in a parallel manner by the aWniun uf dcxanwthasunc to the culture medium. This eflect is biphasic as tl>< eruyme acttvtttcs arc lirst inhibited and then induced when the contcotd axnentrauun is prugrcsswcly raised. The cunirutd also rrxxlities the AHII activity .xt a yualitaNve ba.is. In the absence uf the conicuid, the enzyme activity is inhibited rn r•uru by u• napluMrllavonc but nnt by mctyrapnnc. Dexameth:.xme alsu nKKlthes thc induction of AH) I by PB and BA. both on a qualitative and yuarrutative basu. Ovtrall, un the basts ul' Ihis and other evirknce, it xctns yuuc reaxmabk to assumc that prtnary fetal r:u liver cells tn.ulture might cunstitutc un interesting uwxkl lur studying the physruNppr cal regulatory rncchanunu of drug nxtabulizing enzymes. Girlta. J. t.'% rr ul. In: Snyder rr u( (cd.. ): Htulugrrul Rrurrivr lnrnmrdturrs • ll. Purr A, N. w Yurk: 1'knunt Publuhtng Corp.. 1982. pp. g7-97. Other support: Frnx(s National dc la Rcchcrche Screntrliyue. Protn the Lal>,or.uuuc rk Clwntc Mrdtcalc et Jc Tuxtca,lugte, Inuitut .k P.nMdogn, Uutvtt.dc dk Lt.ge. l.t.tigc. kla'll;tuut. It)
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1 t• MULTIPLICITY OF CYTOCHROME P-450 IN PRIMARY FLTAL HEPATOCY7FS IN CULTURE TM pcrirutal period of life is a critical tiasc for thc quanntauvc and qualitauvc dcvebprncnt of micsusurnal morKwaygcrusscs. and earber ubscrvatiuns suggcst th.d fetal hcpatocytcs in culture might constitute an idcal tuul lur studying the pcnn.tal regufatary mechanism of mooooxygenascs. While it has becn known for a while that pnmary fcul rat hcpatocytcs in culture display diffcrent nwrKxrsygcnasc activiuo which can be induced by several chemical inducers, thcse hcpatucytcs wcrc bclievcd uotil now to produce only one singk cytochrorne P-450 spccies, nanicly the cytu- chrorrre P,rIS0(orP-448). Howevu, it now scems possibic to induce other cynxhrume P-450 spccia in thesc hepatocytes, providing that they receive an appropriate hormu- nal trutmcru. ln the worlt rcportod bers, cxamination was made of the effect of duameQtasooc on various morrooaygenases and on the type of cytochromc P-450 wppatting these enzymic activities. Thrce enzymcs, aryl hydnx:arbun hydruxylasc, ettwaycoumaria doetbylasc and aldnn monooaygcnase, wcre measured for this pur- pose. Results of this study show that the prcsence of dcaamcthasunc in the culturc medium pttoducts qualilative and quantitative changcs in the ownuusygenase-supI^xt- wg cytochrorsre(s) Pr150. For low deaameUusone concentrations, a cytochrumc P-450 is formed displaying biochemical and biophysical propenics simdar to thosc induccd by phenobarbrtat in the adult rat liver. At higher concentratiuns, similar qualitative changes arc observed; but a quantituive phenomenon occurs, the (cynuhrome P-450)- dcpcodeat uszymic activities bciag also induced. Dcxanscthasone also has a syncrgis- tic effoct in the induction of cnzymic activity by the mixturc of p(scnobarbital plus bcrwnthraccae. Tbe various biochemical changes induced by dexamcthawnc in the fetal ccll cultures parallel those observed in vivu during the pennatal period of life. Thcrefore, this ccll culture system may constitute an intcresting model fur studying the onwgcnic development of liver rrwrsooxygcnascs. Krcrsxrs, P.. Goujoa, F., Dc Gracvc, J., Van Cantfon, J. and Gir)rn. J. E. Eruopcan Jo+unal ufBioclremisrry 116:67-72, 1981. OiA.r sswrwt: Foods rk Is Rahen:lte Scicntifiquc Mtrlicalc. From the L..bontoirc ck Chimie Mtdica)e, lnstitut de Pat(wlugie. Untvcrsnt dc Lttgc. LJtgc, Belgium. DNA METIiYLAT1ON IN NORMAL AND SV40-TRANSFORML-D IIUMAN FIBROBLASTS The 5-methykytosine base contcnt of DNA in four numal and (rwr SV4U-(rrnS furmed human diploid fibroblast cullurts was measured by high pcrturnrancc liquid chromatography (HPLC). Rcsults show that the percent uf cytusmes methylatcd fur thc four nonrral ceU lines ranged from 2.83 to 3.18, whrlc thc rangc lur the tuur SV4t1• transforracd cells was from 2.90 to 3.03. The mean (ur thc nNal nuuobcr uf HPLC detcrminations was 2.94 ± 0.28(51 dctcrminatwns) fur thc nurm.l cvll ty(x.:nx13 t10 ± 0.28 (53 dcternuna[ions) for the transformed lines. Thus, in axwa..t to whrr 12 rc{wutu) studics .uatparing normal and oncogcnically transf.uarcd cclls, no appareni dtl lcrcncc was ot»crved in thc 5-rnethyky(osinc to cytusinc base ratius in the two ccll types. It is worth cmplusrzrng that the IIPI.C method used here gives an absolute measurc of the 1)NA bases. In addinun, the purity of the DNA is controlled by urrritunng for thc prescnce uf uracil. Other methods using radioactive label may be hinckrcd by. variuus dtflcrcnt artifacts. Diala, E. S.. Plcnt, M. M., Coalsoa, D. W., and IIu,Q'man, R. M. Biuc)rcmiraf and Biophysical Rrsrarch Carunwsicarions 102(4):1379-1384, 1981. Otbar support: National /nstitutes of Hca)th, The United Cancer Courkil, Inc., TTe Cancer Research Coordinating Committee of the University of California, the Aca- dcmic Senate, University of California, San Diego, and the Leukemia Soctety of Anscrrca. From the Deparunent of Pediatrics, Univcrsity of California at San Diego Schoul uf Mcdicinc, La Jolla. CONSTITUTIVE BEHAVIOR OF METHIONYL-tRNA SYNTHETASE COMPARED TO REPRGSSIBLE BEHAVIOR OF METHIONINE ADENOSYLTRANSFI:RASE IN MAMMAUAN CELLS Methioninc, because of its tules in protein synthesis and in mcthylation, is of central unpurtancc to all cells. In the metabolic prucess, mcthtunmc can be used by the cell through twu dtrfcrcm pathways: (I) nscthioninc can be cunverted to S-aJcrw- syhncthtuninc, the ntajur medtyl source fur cellular uansmcthylation rcacuons ard the source of the prrKrylarninc group for pulyaminc biosynthesa, or (2) it can be convened to nscthiunyl-tRNA, an important inecrmcdiate in protein biusynthesis. In thc paper prcscnted hcrc, it is rcpurted that rnethionyl-tRNA synthccase, unlike rnethioninc ademrsyltransferasc, bchaves in a constitutive manner with respect to the concentratron of methioninc in the culture medium. This behavior is seen in Chinc.c hamster uvary cells and in normal diploid and SV4U-tsansformed hunun 6btublasts. Alahough the kinetics of regulatrun of tncthioninc acknosyltranskrase and rncthiunyl-tRNA synthe- tA.e by cxugenuus rncthiurtinc arc ckatly different, the levels of the two cazymes in the human ccll lines arc similar. Rubnitz, J. E., Jacobscn, S. 1. and Huffman. R. At. Birr htmica tr Bwphystra Acra 677:269-273, 1981. Other sapport: Nauonal Insntuics of Nealth. The United Cancer Cuun.'tl, bk , 71k C'anccr Research Courdinaing Committee of the University ul' California. the A.a- dcmic Senate, Univenrty of Califurnu, San Diego, and the Leukemia S.wrcty of Amcrx a. From the Dcprnn><nt of Pcdratrres. University of CalJornu at San Diego SchcKtl of Muhctnc, La Julla. 13
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FOLATE POLYGLUTAMATE AND MON(X;L(1-fAMA'1'I: AC'C UM(1LA'fIC)N IN NORMAL AND SV4(1TKANSFOKMfiU IIUMAN I7BKO1)LA51:S In the present attcmpt to asccrtain thc role of (utatc palyy.luumalcs in cell divi- siun, it sccmul necessary fint w starvc the cclls of tulatcs uuJ cstnn.nc their total (ulatc requirements for growth. AII four ccll lincs aurdtcd hcrc, the nunnal huu,rn dtpluirl foncskin hbrrsblasl BA, the normal human fctal dipluid AF2. and the SV40-uan.- formod lines P5 and PI, showed similar kinctics lur fulatc starvattun. Scpha.ka O-IQ gel filtratwn chrumatogntphy was used to measure the k-cumulauun of h,latc 1>.dyglu- tunate arKf rranoglutamat.e in all of these cell lincs. After the cells had bcen dcpletcd uf folaru, they were provickd with lirruting anwunts of I'Hl-(uhc acid in order that tlk cells wouW accumulue only forms uf fulate necessary litr pruhfcrauun. Buth the nurmal aod the transformed cells accumulatcd nKmugluumatc and ptolygluwauutc ftxrrrs, but by 72 hours of labeling. the ttansfurn><d cclb cuntairkd 3-Il) unks murc polyglutanutc then the normal cells. Thc growth rates for the tMtnnal atxl lranslunncd ccl(s were similar at this limiting folic acid conccmrauun. Thus, il' (olatc pulygluta- nrates are more important for the prolifcrauun of SV4/1-uanstomxd cclls than thc nrrrnal cells, the inhibition of polyglutarnatc formation could possibly be an tmpurtanl potential target for chemotherapy. llujjnrun, R. M. er ul. Journal of Cellular Physiolugy 109:497-505. 19111. Otltar srpporr: National Institutes of Health, The United Cancei ('riurw•,I, hk'., TIh: Cancer Research Caxdinating Comnuttee of the Univenuy of C'aldorrna, the Aca- dcmic Scnate, University of California. San Diego. arxl the Lcukcmia Society of America. From the f1<partment of Pediatrics, Univcnity of Cahlumu at San Docgo Schuul of Medicine, La Ju(la, and the Genetics Unrt, Children's Scrvtce. Massarhuscas Gcncral fiospital, Department of Pediatrics and Center for Hunun Getx:tic., 1(arvard Medical School. Boston. DNA METHYLATION LEVELS IN NORMAL ANI) CHEMICALLY-TRANSFORMED MOUSE 3T3 CELLS -iltis investigation was undertakcn to assess the effcct uf chcmtcal trnn.funnanun on wul gcrwmic DNA rnethylation as mcasured by high Ixrlunnarxc liquid cluumr- wgr'aphy (HPLC). In the study presented hcrc, nunual nmnrx: cmbryu 3T3 cell cultures and prose oacogcnically transformed by the chemical c:rrcinugcns IxniiNa)pyrcrk aud rncthykholanthrene were analyzed by HPLC tu rktcmmrc the S-nkthycytn.uk to cytosine base ratios in their total gcnumic DNA. Rcsults showed that the nKan hu 10 HPLC dcterminations uf the normal 3T3 cells was 2.1/TJ6 of cytusmes nuahylatcd with a standard dcviation of s0.S8, while thal for the bcntt(a)pyrcnc-tran.funucd 31:3 cells and the methykhufanthrene-transformed 3T3 cells was 2.It11'i• s U. 31 113 rktcrmt- nationsl and 2.81'b 20.18 (IS determinatruns), respecUvcly. These rc.ults led tu thc conclusirm that there is no real diffcrcnce in the catcnt of wtal gcnonu. UNA rnw•ohyla- twn between normal and chcmically-uansformcd 3T3 cells whcn mcawrcd by I IPL('. 1)iala, (:. S. and lluf/nwn, R. M. C11z lLiwhrtnicul und llmphysirul Rr.tnurh Cummunirutrun.t 1(ll(4/:(4K9-14'N, 1482. ~ Other support: National Insuwtcs of Ucalth, The Unitcd Cancer Cuuncil, Inc.. lbe T„I (']nlCr Kcxuch C00ndInating Cumnuttcc of the Univcnny u( Cahhnnta. the Aca- rkm,c Scnatc. University of California. San Dicgu, and the Lcukcmia Society o( Anknca. Prum tlw lhpartn>Lnt uf Ncdiatrics. University of Calilurma at San Diego School of Mcdkutc, La Jolla. M6f111UNINE DEPENDENCE IN CANCER CELLS-A REVIEW Mcthir>,unc dcpcrukncc uccun tn a largc numher and wide variety u( canccr cells and dr>LS not xcm w be a random cumpuncnt of the transfurrncd phenotype. Dchnruun ol' methiuninc rkpcr>d<.nce states that it is a defect found in many cancer ccll hncs that inhibits their growth in culture when methionine is replxed by its imrncdiate precur- sur, humr><ystcine, in the culture medium. Normal cultured cell. do nut have thts dcfcrt. Tlris reprxt lists the diverse and large number of anunal and human cancer lines that arc mcthiuninc-dcperwknt, and critically reviews the ccll biology and mclhiomne birxhcnusuy of the phenomenon. 11u,0'mun, R. M. In Vitru 18(5):421-428, 1982. Olher support: National Inatitutcs of Hcalth, The United Cancer Council. Inc.. The Canrer Keacarch Cr>,ndtnaung Crxtunutce of the Univcrsity af C.rltkxnu, the Aca- demic Senate. Univcnity of Caldrunia, San Diego. anrl the (.cukemia Society of America. prruo Uw lkpanuwm of ('cdrautcs. Unrvcrsuy of Cahlurma at San Diego S:huul of Mcd,ctna, la Jolla. IIYI'OMETIIYI.ATION OF 11i:LA CELL DNA AND THE ABSENCF. UF 5 MI~fHYL(.'YTOSINE IN SV4U AND ADENOVIRUS (TYPE 2) DNA: ANALYSIS t)Y 111'LC In thc study presented hcrc, nxthylatiun of the punhcd viri.nt 1)NA uf b.,th SV4U arKl .nlcnrrvirus (typc 2) wa. n>,'asurcd by high pcA,unt.rncc liquid c1u,MU.rtuKr.rphy (IIPI,(') :url crnulwcd to tlkrr Mx.ts. Afrk:rn gr.rn ru,wtl.ry kidney ccllk and IkL-A cclls, rca>lcuvcly. In SV4O DNA, as nwch as 12 na,MHmrks ul cytusmc has brcn n,.a.urr:d without crNrcumit.tnt rkt.ctn,n of mCyt. $V40 iuruarns 27 C'p(i parnm which ra tlk u.u.rl m.thylabuu sttc. II all Cp(i (t.rr% were nrcthylitcd. tho wuuW ytcW a srgn,bcanr 1.3% rr>Lthylatron uf tutal cytustncs; Mrwcvcr, ut the vtnun DNA studt.d here, nuru sccnKd tu bc n><thylatcd. Alsu, as with SV4U, m'C yt was mM present in the 15 14
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ekµioa patscrn of virion adcnovinu (type 2) DNA basea. Ovcrall, essentially no 5- methykytosine was ducctod in cithcr viral DNA. lmplicatiuns (ur viral gcnc rcgula- lion by metAylatioa are discusscd herc. In comparison with normal human ccll DNA mct(rylatioa kvels, liel.a ccll DNA mcthyluion is rc.luccd stgntficantly. Diala, E. S. and !/o,Q'man. R. M. Biochemical and Biophysical Rcsearch Communicutions 107(l):19-26, 1982. Olkar sstipport: Natioaal IrWitutu of Health. From the Dcpuvncat of Pediatrics, Univcrsity of California at San Dicgu School of Medicine. La Jolla. REDUCED AVAILABIUTY OF ENDOGENOUSLY SYNTIif:Sl"LI:D METHIONINE FOR S-ADENOSYIvfEiH1ONINE FORMATION IN METHIONINE-DEPF1dDFNT CANCER CELLS Mcthioninc (Mcl) dcpendcncc-i.a, the inability of culturcd cells to grow when Met is replaccd by its irrunodiate precursor hornocystcme (Met- Hcy' mca(wm)-is a frequent component of the oncogcnically transformcd phcnotype. Normal cclls, on the other hand, grpw in this rnodium. Thcnc have been reports 1Hof(man, R. M. & Erbc. R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527; Hoffman. R. M., Jacobsen, S. J. dc Erbc, R. W. (1978)Biochcm. Biophys. Res. Commun. 82, 225-2341 of normal or Aighcs rates of Met biosynthcsis in Met-dcpcrrdem cells and a postulation that Mct- dcpcndent cclls are dcficicnt in utilization of cadogenously synthcsrud Met as opposed to cxogerwusly supplied Met. To rutswcr the critical question of what biochcmical rcauion(s) requires prcfornrod Met in Mcl-depcndcnt cells, we labclcd cells with Mct- troe IS1 i{cy or ('S) Met and dctcnnincd the kvels of Met. S-sdenosyhoetluunine (AdoMU), and S-sdenosyRromocysteine (AdoHcy). We report hcre cxpcrinrents that dcasonsocare that Met-dcpcrsdcal cclls synthesizc a rxxnul arrxwnt of cndugcmwaly syatDcsizod Met and are deficient in utilizing this Met for AduMu synthesis. In cootraat, exogcrsously suppliod Met is utilized normally for AduMct brusynthcsrs. The ratio of AdoMet to AdoHcy is low in Mcldcpcndent cclls growing in Mct'tlcy' nrodium. Wcducrminod that the low AdoMct/AdoHcy ratro probably lirnita growth of Met-0epceduu cells in Mct-Hcy• medium. Coa)son, D. W., Mccham, J. 0.. Stern, P. H., and IloQman, R. M. Proceedings of the National Acadenry of Sciences of the Unired Srutes of America 79:4248-4251, 1982. O(Jssr srryport: Natioasl Institutes of Hca)th, The Univerauy Cancer Cuunctl, Inc.. The Cancer Research Coordinating Cornmrttcc of the Univcnity of California, the Acadcmic Senate. UNvctsrty of CaJtfornrs at San DKgo. and the Lcukemra Society of America. From the Departnsent of Pcdiatncs. University of California uat San Diego Schr«d of Modiciac, La Jolla. VASCULAR INVASION OF CARTILAGE: CORRELATION OF MOKPHOLOGY WITH LYSOZYME, GLYCOSAMINOGLY('ANS, PKOTEASE, AND PROTEASE-INHllI1TORY ACTIVITY DURING IiNDOCIIONURAL BONE DEVELOPMENT Although it is well known that cartilage vasculanzation is a prercquuitc fur txxte dtllcrcntiation, tlrc prcasc mechanisms of this vascularizatwn arc incompletely wxlcr- strxrJ. In the ptcscnt attcmpt to study these changes. dcmincralrzcd bone matrix prc- parul front rat diaphyacs was trsnsplanted subcutaneously into bilateral sites in the thoracic region of 28- to 35-day old malc Long-Evam strain rats; the day of transplan- uuon was dcsignateJ as Day 0. While using the rnatrix-induced endrkhundnl bone ddfcrenuaticrn as a rnoJel systcm, changes in the kvcls of lysozymc, patterns of glycosarnirurglycans, and actrvitics of protcascs and protcase inhibitors were studted during matrix-induced carrilage, bone, and buttc marrow development The morpbo- hrgtcal transitions were corrclatcul with the biochemical parameters. Rcsults showed that thcre was a pcak in lywzynse content on Day 3. during mcsenchymal cell prolifcr- auun, (ulluwed by a decline during cndochorx)rsl bone formation. The lysozymc levels increased again and attained masimal values during hcmatopoicsis on Day 21. Pro- tcasc-inhibitory activity was maximal during chorxLogencsis and drnunishcd during ostcogcncsis. Prutcase activity was maximal on Day 3 during mescnchymal ccll prolrf- «auon and was apparcntly prcscnt as an enzynre-inhibitor complex. Vasculanzatron and bone formation were accompanied by an increase in protcase activity. Chon- druitin-4-sulfate was the prcdonunant glycosaminoglycan detected in the nutrix-in- duccd cartilagc and bone. Reddi, A. 11. and Kueuner, K. E. Developmentul Biology 82:217-223, 1981. Other supporr: National Institutes of Hcalth. From the Laboratory of Biological Structure, National Institute of fkntal Rcsearch, National Institutes of Health. Bcthexla, MD., and the Depanmcnts of Onhopedic Surgery and Ihochetnrstry, Rush-Presbyterian-St. Luhc's Medreal Ccntcr, Chicago. RESISTANCE OF CARTILAGG TO INVASION In this cxtensrve and cucfully workcd-out chaptcr, a simplified conccpt of the invaaivc procc» is intruduccd, and a unique sequence of cvcnts occurring during physiologic (emkxhelial cells) and paehologic (ostcosarconu cclls) invasion is dcscnbuf. The term invasion, as used here, is dchncd as the pa»rng, rntcrpencuatrun, of infiltration of cells into ad)acent tissues. The events that characterize this prrrccss have been studicd in vuro in a combincd organ-ccll culture syucm consisting of marnalian hyalinc cartilage and TE-85 osteosarcoma cells. Cartilage was ab-e to reaist the invasion by uateusarconu cclls in the organ-cell culture system. Ostarsaramu cells forced to grow in direct contact with the cut surfaces of articular hyalrnc cartilage pikd up to sevcral cell layers, but were unable to penetrate the canilagmous nutnx. In vivo. similar reatrictiona of cantlagc to tumot cell invasion havc been rc(xxtcd in ptinury and n><tastalic tumors. Also, cartilage is thought to be resprmatbk for the slow invasion of carcrnuma cells into the walls of the larynx. Spccihc studies reprwred here shuw that hyilrnc canilagc, mddly cxuarted with vatiuus salt auluriuna, is readily ieGltrated by uateuaarcunu cells in the urgan-eell culture system. These lmdmgs kd to 16 17
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th.: conclusion thar canilagc cuntatns caraelahlc matrrs .anqxrunJs that inlnhn inv.r- rc sNm in an e apenmental system. W frcn these Ji((usahlc and c s tra.-tahtc subslarrccs wC further studicd. it was found that the inhibition ul ostcrrsarconra cell pruhfcnuon was caused by nwkcuks with a molecular weight of less than 50.1(X) Jaltotu. From thu anti-invuive factux of the canilage extract, a prutcasc inluhitur was irknNlic.l that has tbe ability tu inhibit nuntalian collagcnast, InCludlnl7 that CIaMnBICJ by UNtCoAarlUnra cells And errdWlwlial cclls. These espcrmwntal data led ar tlrc hylartlresrs that rnv.rsrun of citlx:r wrnrx tx endothchal cells depcnds on pruteulyu.• (<nll.rgenulyuc) cniyme xtivities. Other studies along these hrres havc Ixen in.ntutcJ tu lidluw the rnvasrvc- ncss and proliferation of bladder carkcr. Krurtnar, K. E. And Pauli, fl. U. In: Gilben. ff. A., Wciss, L. and Monscn, D. C G. /eJs•>: Rrrnr Alrtu.rru.rr.r, lsustom: G. K. Hall Medical Publishcrs, 1981, pp. 131-Ib5. Otbsr srrppat: National lnstitutes uf Health. From the [kpartrnents of Orthopedic Surgcry, Pathology and l)io<Ikmutry, Rush- Presbytcnan-St. Lure's Medical Centcr, Chicago. ANTIINVASION FACTOR MEDIATES AVASCULARITY 01: IIYALINE CARTILAGE To test an aruiinvasiun (actor (AIF) hyputlxxsis uf Q>t resutarwc u( canilag. to vascular rnvrsion, a novel in vitro system was surdicd that cmpluycd twvuu ua: ular canilage as a growth surface for normal hcparin-stimulatcd endrxlxbal cells. In (hrs study, cells were tested for their ability to invade the matns uf viable urd Jcvitalv.cJ extracted cartilage as monitored by thin-sectwn ckctron mtcruscupy. The growth bchavior of cells on rkvitalized extracted cartilage was cxamirred in the prcxnce anJ abserrce of cartilage-dcrived, extractabk AIF in the culture medrum. Whereas nor- mally viable anictrlu cartilage is a poor growth surface for crrdutluhal cdls, the eodothelial eells studied here, in contrast, grew as cuntact-inhrbitcd nwnulaycn u( flattcned cells on the surfaces of extracted cartilage. The cells were +cparatcJ Iruur the cartilage matrix by rrbundant basal lamina. lbcrc were a few rnicruvilli at the basrl plasma merrrbrane, but thcrc was no dcgradation or pcrutr.nun u( the cullagcnuus mauix uf extracted cartilage. Huwtver, when endutfxlral cells were sunrulatcd by heparin, they aswmtd a polyhedral shape and perktratcu thc extrxtcJ canilagc wu;r numerous mierovilli and some cytoplasmic processes. Thrs penetration ul tfic tulla- gcnous rrsasna was associated with tissue rarclaction and degradation ol collagen fibcrs. lnrportandy. howevcr, this invasion of hcparrn-stunulatcd a>,kdhclral cells wns abolishe.J when low concentrations of canilagc-deriveJ AIF werc added tu tik culrurc medium. These data provide evidence that the resrsurrKC uf hyahrrc cartilage to cn- duthclial cell invasion is regulated in pan by tissue JenvcJ prutcin+ac rnhrbuurs anJ an arstiproli(erative activity direeted against errduthehal cclls. Krrrrtrrtr. K.E. er d Scnrinurs in Arthritis 6 Rhtrurwpsrrr 11:67-69, 19Z<1. Ot/ur srroPort: National Institutes of lkalth. From Rush Medreal Culkgc. Chicago. 1>S CUAKACTIiKI-LATION OF ADULT BOVINE ARTICULAK CliONUROCYT(iS IN CUL'1-UKE Anrs:ular cartilage sliccs, obtaincJ from 1g-mrr-ulrl brrvrnc metacarprsphalangcal juints, were used as source nutenal (or this rkscnprive articlc ol eMrnduxytes in culture. A(tcr sequential digestion and hlu.tion, .clls wcrc plated in either nssue cuhure Jrshcs or ruller IxNtks. ChurrJrucytes fixed in bulfcrcd glutarakkhyde corrtain- ing 0.1% rullKnlum rcJ, were examined by light And transmr.sn>rr electron mkru- srupy. Cullagcn lype dclerrninalron of 7l-prohnc-labekd pnrtcins iwlated frunt culrures were IvrlurnkJ by clcctropMxctic and CN(3r pcpridc analyso. (Siu.ynnccsis of protcuglycrns was nrcasured by "JO, rncurporatrun into macromulccuks cxtrxtcJ under drssur:iauvc conditions. Eaaminatton showed that iwlatcJ chundrucytc. prior to culture were typically rounded with scanl territorial matrra, which could be removed by mild trypsiniiation. Throughtwt the progressiun of the cultures, phcnutypw alkra- buns were mN observed. Ekctrofluorugraphs of collagen tfut was exxracted Irrxn I f- prulhnc-labclcd cultures after mild pepsin digestion showed I band in the position of the aI chain; an a2 chain could rrot be detected. Cyanogcn bromide pcplidc analysrs confimxd that the majur radioactive peptides eornigrated with unlabeled peptides obtained from type 11 collagen- type I collagcn was nut dctectabk in these . ultures. Prutcoglycan aggrcgatc was extracted from both culture Jishcs And roller bottle cul- tures, under associative conditions. There were dif(ererrres noted in this cxpcnment suggestmg that the roller culture ccll-associatcJ matrix may hase a greater rkgree of txganization than that grown in standard tissue culture drshcs. Thcse samc data also indicate that articular chorwlrocytcs grown in mass rullcrcultures are capabk of synthc- sizing a plremrtypically stable, tiuuc-like matrix in viuo. Kwttrner. K. E. tr ul. Srmrnun in Anhrrus 6 Rhtumurisnr 11:101-103, 198 1. Odur supporr: National Institutes of Health. From Rush Mcdical College and the University of Illinur. Dental S:h.w1, Chrcagu. KL•GUI.ATIUN OF TUMOR INVASION BY CAKTILAGE-t)ERIVED ANTI-INVASIUN FACTOR IN VITRO Mamrnaltan candagc is highly resistant to invasion by tunwr irll. TTrr. reast- arkc was studied hcrc with the usc uf a novel in vurr.:ullure sysrcm. Anrculu:anilagc obtained Irum Iresh nrctacarlwrtralangcal jornts ul prcaJrrlcsarnt twvuks was uxda. a gnrwth surfacc lrrr huuran TE-g5 ustcusarcoma cclls and hrreskrn hbrubla.ts. ('artrlagc drsAs furmeJ t1k bullunn of stainkss-sleel cylrndera, providing cluscJ growth charn- hrrs for these cells. Hnlh invasive ostcosarcuma rclls And normal hbrubla.ts were unable tu pcnruatc vobl., uneatracted canilage during a 2-wecl cuhure pcrMrJ Wlno cartilage was JcvntalucJ by lrcctrng arxd thawrng, the trssue renr.rrncJ resutanl to invasion Cutrlagc, extractcJ with either I or 3 M gu.worJrnc hydnMhkrrrde, was invar)eJ by ost.usarcrtina cells. but nrM by control bbruMasu. Inva.arn by usteosar- cunra cclls rnlu salt catrx'tcJ cartdagc was ahuhslrcJ wlrrn low c.xr.•rnpatnrns u( a cartrlage-dknveJ, anu; rnvasaxr (actor were adrkd to the culture nreJrunr. These data 19 :'3
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!h pruvided evidcncc that the rcsiatanic of carUlagc to tunr.x invasion is rcgulatcd in part by lissue-dcnved prutcinasc inhibiton. Pauli, B. U., Memoli, V. A. and Kwrrrncr, K. E. Jownal of rhe National Cancer lnsrirwu 67(1):65-73, 1981. Odwr suPport: National Canccr lrutitutc. From the Departments of Pat)rology. Biochcmnuy and Orthupcdic Surgery. Kush MedicaJ Culkge, Rtub-Prabytuian-St. Luke's Medical Center, Chicago. lN VlfRO Dlif1:RMINATION OF TUMOR 1NVASIVI:NLSS USING EXTRAC7ID NYALNE CARTILAGE In this attempt to determine whether salt-extracted cartilage could be used as a,est connective tissue for in vitro discnmination between rroninvasive and invasivc tunwr cell lines, a novel in vitro method was devised which used salt-c.uactcd, bovine articular cartilage as an experimental growth surface for bbuh nonnal bladder cpithclial cells and noninvasive, invuive, and Inetastahc carclnuma cell lines derived frUnl chemical carcurogen-induced tumors of the rat urinary bladder. As nwntwrcd by thin- section electron microscopy, salt-exuacled cartilage was readily pcnetrated by the invasive and metastuic rat bladder carcinuma cell lines. The mctastatic ccll line could be diffaentiated from the invasive, nunmcustauc cell line by its greater depth of invasion. In congrast, nrxrinvasive carcinoma cells as well as normal bladd.:r epithchal cells lacked the capacity to erode and penetrate thc extracted nratrix of the anrcular cartilage. Using these defined cell lincs, sah-extracted carulage can be used to rcprudu- cibly discnminate between carcinomas having different mvastve pwemials. This assay system may have diagnostic application for the in vitro staging of tumors. Pauli, B. U., Memoli, V. A. arrd Kurttncr, K. E. Cancer Research 41:2084-2091, 1981. OtMsr suyporY: National Institutes of Health. From the Deputments of Pathology. Biochemistry, aud Orthulrcncc Surgcry. Ku.h Medical College and Kush Culkge of Hea)th Sciaucs, Kush-Reshytcrun-St. Lukc's Medical Centcr, Chicago. A FAMILIAL AGGREGATION OF PANCREATIC CANCER: AN IN VITKO STUDY Pancreatic cancer, with its obscure etiology and drfhcult carly dugnusu, prescuts a medical problem of staggenng proportions. In the in viau study prcxnted hcrc, a family was ideruifred in which four individuals manrksted pancreatic can.cr venhcd thrrxrgh two generations. Cell cultures from spbl-lhrd,ness skin brupsy .Ikcurxns were obtained for 24 members from three generatrons (17 blurxllrnc relanvcs, scvcn Iamdy members by marriage) as well as ten nonfamJy rw,rnul sublccts. nunc wnh a family histury of solid tunrors. One of the constant fcatures of hurnan tunwlrycr dcrmal cultures has been diploidy. On the other hand, hypcrdtplutdy, uthcr than 20 I I tctrapluidy, has been rarely observed in cultures from normal subjects without a family history of solid tunwxs. In this study, howcver, increased in vrrru hypcrdrpluidy was observed in eight of the 17 family rnembers tested. In the long run, aggregates of pancreatic canccr in families such as this one, and the occurrence of pancreatic carner in surne autusumal dominant cancer syndromes have added credence to the relevancy of a gcnctic cumponcnt(s) in a fraction of pancreatic cancers. In an imprxunt way, this study has deriMrnstrated the putential for combining detailed fanuly data with recogni- uun of in vitro brunrarkers for carker proneness as an approxh to the comprehension of carcinugcncsis in pancreatic cancer. Danes, Il. S. arKl Lynrh, Il. T. JAMA 247(2u):2798-28U2. 1982. Otticr suyporr: National Institutes of I lealth, Dartes Medical Research Fund. Corncll University Mcdreal Cullcgc, and Zemurray Foundation. From lhe Laboratory for Cell Biology. Department of Medicine, Crxnell University Colkge, New York, and the Department of Preventive Mcdicine/Public Health. Crcighton University School of Medicine. Omaha. GENL'fIC/EPIDfiM1OLOGICAL FINDINGS IN A STUDY OF SMOKING-ASSOCIATf:D TUMORS In this gcnctic/cpidcmiulogical study, family histories of canccr were uhtained via pcrxmal interviews from consecutively ascertained canccr pancnts wlw wcrc under cvaluattun in unc of two University Oncology Clinics in Nebraska. Included in the xncs were 147 breast cancer prubands, 85 colon cancer probands, 88 lung carrcer prubarrds, and I I I probands with uthcr canccrs that have bccn rcpunnc.l tu bc asxsciatcd with cigarette smoking (carcinoma of the oral cavtty, esophagus, pancrcas and urinary bladder). Smoking histurics of prubands and their relatives were ubtarncd fur an overlapping scncs of 60 lung cancer probands and 78 pnrbands wirlr other snaking- asxKialed tuonrrs. Findings revealed that, although a stgndreant cohort ellect was ubserved with respect to smokmg habits fur both relatives of lung cancer probuwk and (ur relatives of Ixubands with other snruking-assuciated tumors, a corresponding trend lur lung cancer Ireyucncy was observed only for rclatives of lung cancer probands. 11m, result suggests the importance uf host tactors in combination with environmental cspusures in detcmrming lung nsk. A cuhurt trend for lung canccr was also apparenl anwmg relatives of breast cancer prvbarrls, but not for rclalivcs uf colon can.'er pnr- barkJs, suggcsung the possibility of an intrinsic association between carcmurrus of the brcast and lung. It scems reasunabk that further elucxLalun of host laraa suss:cp/tMb ity in lung canrcr may have irrrprxtuu etrulogical and preventive tutplKatrons. l.ynrh. !l. T. rr ul. C'unrrr Grnrnrs unJ O9ugrnrrurs 6:163-169, 1982. t•rrNn thc Insrawtc lur Fanulral Cancer Managcrncnt arwl Control. Inc.. Ikpannknu of Prcvcnnvc Mcditine/1'ubbc fkalth, Surgery. and Pathulugy, Creighton Untversuy Schuul uf Medicine, Onuha. 2I
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r LOW SERUM I8A IN A FAMILIAL OVARIAN CANCER AGGREGATE AlUwugh familial ovarian cancer is irkrcasingly rccognt[cd. cun.utcnt biomartrcr acaociatiops that correlate with its risk have rcmaincJ elusive. In Ihi, paper. however, a family is describcd that is charactcrized by excessivc t><currcnces uf ovarian carcinoma tsanr:miUed in a pattern consis(cnl with an aulusomal duminaut factor. This family is unique in that identical twin sisters cach had vcrilicJ ovarian caoccr and each had a daughlcr with the same ksion. Upon famihal invcstigauun, 14 of 45 individua)s tested has serum IgA kvcls bcbw Ux 95%, range. Carcful cunsukntrun of these results showorl that low serum IgA kvels wcrc found to scgrcgatc in a wfticicnl number of individuals from this family tu suggcst that thb may bc a gcncu- cally ddermined immune defect ctiologically integral to carw•cr suscc{xrbtlity. While the putative role of IgA in pathogencsis rcmains elusive sull, tt secros apparcnt that carsr.er-prorre familia should be Ihoruughly investigatcd frx funhcr cluctJatiun ul these phenomena. Sclrucikc, G. S., Lynch. !/. T.. Lynch, J. F., Fain, I'. R., and Chalxrun, li. A. Cancer Genctics and Cytogcrsetics 6:231:236, 19g2. Orlsr suppwt: Elsa U. Pardee Foundation. From the Departrnents of Medical Microbiology and Prcvcnttve Mcdreutc/Publtc Health, Crcighton University School of Medicine, Omaha. NATURAL HISTORY OF HEREDITARY CANCER OF TI I[: BRt:AST ANu COLON Anecdotal rrports have suggested that survival charactcrisucs of hcreditary 1'uruts of cancer may differ from their sporadic counterparts. To test this possibility, a rcvicw of disease-froe survival was undertaken by evaluating the affected mcmbers ul' hercdi- tary colon and breast cancer families within the extensivc Crcighton University Oruul- ogy Center familial cancer resources. In the study presented here, the natural history of 106 patients from 18 familiu manifesting hereditary brcast canccr syndrunxs and uf 117 affected patierus from 20 families manifesting nonpulypusis hereditary colon can- ea was evaluated. Findings werc compared with the Amcrican College of Surgcuns (ACS) long-terra audits for breast and colon cancer respecttvely. The cardinal features of huoditary cancer were observed within the study group, including: (1) a signiticant youaaer age of onxt (49 years, brcast; 46 years, colon); (2) an exccss of prusirrul lesions io the Aereditary colon serics (49%); and (3) an cxcess of btlatcrahty in the herodiury brcast cancer patienls.'Rre clinical stage at presentatir>,t was similar fur tlk hcrodiury and ACS audit patients. Five-year survival was signdicantly irnpruveJ 1ur both hereditary canecr populatiau as compared to the ACS audus (67')6 hcrcdit.rry boeast cancer assd 52% noopolyposis hereditary colon cancer). Irupruved survival rn hereditary eobn aad breast cancer patients may have a bearing on the design uf future clinica) protocols. Albano, W. A., Rocabancn, J. A.. Lynch, Jl. T.. Canrpbell, A. S., MuIItuJ. J. A., Orgw, C. H.. Lynefr, J. F., and Kimberling, W. J. Cancer SU(2):360-369, 1982. OrJKr support: National Cancer Instrtute. From the Institute for Familial Cancer Managcrnent and Cunuul, Inc.. Crcighton University School of Medicirse, Omaha. 22 NIiUPI.ASI'1("1'RANSFURMA'IION OF RA!)BIT CELLS BY MURINE ~ SARCOMA VIRUSES lfj Whik: rabbit cells have been widely used for is)lation and rcphcaurur of a vancty ~ ul rctruvirusc., they have sckfom been used fur lransfurmation studies with murine T i sarcuma viru.cs (MSV). In this paper, however, ncopla.tic tramfurmatiun uf rabbit cclls by Kintcn MSV (Ki-MSV), the Ki-MSV pscudotypc ul' baboon enJugerwus virus (KirMSV IIialiV/) arrd the Muluncy-MSV pscudWype ol fclinc Icukcmu virus (M-MSV IFcI.V 1) t% rclxKtcd. Rabbit cclls can be readily uanslurnkJ by KiMSV. Ki- MS V( IIaGV ) and M-MS V( FcLV ). Rabbit cclls transforrucJ by KrMSV rnJ M-MS V- lFc1.V) were IuunJ to be virus prax)uccrs, whcrcu those uans(urnrcd by KrMSV(I)aliV) wcre nrmlxrH)ucen INP). The NP cell% were obtained by simply ~ mlccung rabbit cclls with Ki-MSV(B.EV) and wbcultunng the rn(cctcJ cells. AI- ~ tl>,wgh ghc nMrrldnduktcally akcreJ NI' cclls did not pn>t)uce infccturus vtru. ur nwnne ~ Icukcmu viru. anUgcn, dw:y did contain a rescuablc MSV gerwtme All of dK' trun- F, . IurnrcJ cclfs lurnkd cukmres in $ult agar, grew tu high salurauun densities and pru- Juccd tunwn when Iran.planlcd into nude mice. This report is thc hnt one of U succcssful tuaNrr induiuun by vuus-transforrned rabbit cells. The abtltty lu produce sarcomas in rabbits by KrMSV and M-MSV(FeLV)•transhxmed cclls.hould provide a useful additrunal mrxlcl for studying chenw/hmmunotherapy as well as immuno- prevention of rabbit canccrs. Rhirn, J. S., Bcdigun, If. G. and FoA, R. R. (Meirr, Jl.) JnrrrnuriunalJuurnul uf'Cunrrr 3U:365-369, 1982. Other support: Nauunal Science FounJauun and the National Insntutes of Health. From the Lab+xatury uf Cellular and Molecular Btulogy, National Cancer Institute, I)cthesda, MD. anJ The lackson Laburatory, Bar Harbor. ME. 11. The Respiratory System Ii1.ASTIN IIICriYN'1111iSIS IN ('/1ICK EMBRYONIC LUNG TISSUE. COMPARISON lY) CHICK AORTIC ELASTIN In Ihrs M.xhcnuc:d study, the synthesis of clasnn wAs lulluwcJ rn chick cmbry- unrc lung and cuatpucd tu that seen in enrbrylMN aUrtlc tissue. MCssCn(Yr rIl1,N1ucICN a.rJ (mRNA) was uul:rtcJ frum both lung and aonic huucs anJ Ir:ursl.r.J in an niRNA-rkperxknl nbbu rcticulucyte lysate. The results demurrstratc that brxh U»uc RNA prcparauun. Jrrcct Urr synQrau of two clastrn pnucrns pr»xssmg nw.kcuLrr weights ut 7U (Nri) and 73 (XX), which are tmmunuprcripnable with anuborly JtrectcJ rgaina cluck :xuuc trupucl.rstin. ( hg:rn: ulture u/ embryrrmc lungs anJ xrrtas Iu11uweJ by extraruuo ul t)re 1'llIvalrnc-labclcd proteins with urea in the presence ul rcduitng 23
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msd alkylating rugcnts rcvcakd thc presence of two immunorcactivc clastin pro(cins similar to those synthesized in the cell-frec systcm. Limttcd tryptic and chymutryptic pcptidc mapping of die two elastin proteins synthcsized in aortic organ cultures re- vcak4 a strong homology buwetn the proteins with only minor dctcctabic dif fcrenccs. Automated sequencing of ('H)valinc-labcled tropoclastin 70 000-dalton species) iso- lated from aorta artd lung organ cultures demonstrated idcntical positions of vahnc rcsiduts in thc NH; tcrnrirral rcgioa of both proteins. These results dcmtuutraue that elastin synthesis in two unique embryonic tissues involvca thc production of two distinct polypcptide chains, referred to in this paper as tropoclastin a and b. Also, the 70 OOOdalton protcin (trupoclastin b) is similar in ekctroptwrctic behavior to convcn- tiorul tropoelrutin and appean to be idaNical in aortic and lung tusucs as juJgcd by anrirro acid analyses, c)edrophorctic nsigration, high-pressure h(juid ciuurnatography, and auuxrwcd aoqucact analyses. Fosur, J. A. at o1. BiucAcnrisrry 20(12):3528-3535, 1981. OtAsrsrrpporC Natiorra) Institutcs of Iicalth and die National Ftwrxlatwn March of Dimca. From the Dcparurscat of Biochemisuy, University of Georgia, Athcns. DIFFERENTIAL EXPRESSION OF AORTIC AND LUNG ELASTIN GENES DURING CH1CK EMBRYOGENESIS This quaotitative study addresses the question of whcther or not the ratio of trupoelastin b to a changes during cmbryonic dcvclopmcnt. To mcasurc thrs, the rates of tropoclastin a and b synthesis were determincd in shon-tcrm organ culturc. The results dcrranstralcd that in lung tissue the ratio of the two trupoclastins remained essentially constant. Each of the tropcxlastins cornpriscd 50% of the total clastm synthesis. In the aoctic tissuc, however, tropoclastin b represented 7U% of the uxal clauin in the 11- to 13 day embryos arsd incrcascd to 91`b by Day 16. Thcsc ubservatiuns socn in the organ culture system were paralkled in mcasurcmcnts of fwxuunal mRNAs coding for dre two proteins. Mcasuremcnts uf functionnal tnopuclastin nrRNAs coding for the two proteins. Measurements of functional tropoclastin mKNAs from both lung and aortic tissues were performed in an mRNA-ckpendcnt rabbit rcticulocytc lysate system. Although the changes in the abundance of the «opuelasun mKNAs rcvealed the same tttnd as that seen in the organ culture data, the nugnitudc of OK tropoclastia b to a ratio in the aortic culture was twice thar dctcmrined in the cell-frce translation of aortic mRNAs. The most imponant uutcwnc uf this study was the howhnl; that the ratios of the ratu of synthesis of uopoclastin b to a u well as the anKwnt uf functional mRNA corliag for these proteins differ with development as well as with tissue. Speci6cally, the data obtained frorn both cell-frce translauuns u>,l organ culture capuimcnts dcmorrstrate that therc is a differential cspression of clasun gcncs dunng snna developrncat which is significantly different from that found in developing lung. Bamncau, L. L., Rich, C. B., Przybyla, A. and Fusser, J. A. Devsloprwanrd 8iolop 87:46-51, 1981. Wisr srprort: NatiauJ Institutes of Health and the Natrunal F.wnJattun Marih of Dimcs. From the Dcpartment of Biochemistry. University of Gcurgu, Athcns. A SURVEY OF SENSITIVE TECHNIQUES FOR EXAMINING THE lN VITRO SYNTHESiS OF ELASTIN Five sensitive techmqucs for identifscation, quantiution, and chuactcnzation of soluble clastin increases arc described and elaboratuf upon in this paper. All of these mcUrotls are applicable to picomolar amounts of tropoelastin generally encountered in in vitro systems. Thert techniques uc: 2-Dimcnsional Gcl Ekctroplrorests, Quantita- tiar of Tropoclastin, lmmunoprecipitation, Micrrnequcncing of !n Vuru Labeled Tro- poclauin, and Pcptide Mapping. The trxhniqucs described here provide the necessary sensitivity fpr dcscribing, buqr direcUy and irdirectly, the presence of oropoclastrns synJrestzed in in vrtru systems. Fusur, J. A. rt aI. CG»rnectivs Tusut Rcsewc6 8:259-262, 1981. Orlur support: National Institutes of f kalth. Frwrr die Dcpartment of Biochemistry, University of Gcorgia, Athens. EVIDENCE FOR IN VIVO INTERNALIZATION OF HUMAN LEUKOCYTE ELASTASE BY ALVEOLAR MACROPHAGES Although imcraction with alpha; protcinasc inhibitor (n,Pl) is considered to be the major mcuhamsm for the inactivation and ckarance of potentially harmful pro- tcases, such as kulcocytc clastase, from the lung, recent evidence has suggcsted that direct binding and internalization of human kulcocyte clastasc (HLE) by alveolar macrophagcs may rcprescM an alternative route for the inactivatrun and elimination of this lung-danraging protcasc. In the work reported herc, cell lysates from cultured human alveolar macrophagcs, dcrived from the lungs of cigarette smokers, were shown to contain detectable amounts of aa elastinolytic cnzyme. Although paruculatc clastin was solubilizcd only after prolonged incubations, lysares readily hydrolyzed t- BOC-alanyl-p-niuophcnol-cster. Hydrolysis of thc latter substrate was inhibited by the kurocyte clastase stte-spcci6c inhibuor. N-ac-(ala),-chlorumcthyl kctonc. In addi- tirNr, r adwnnmurrr><kcurl+(xM esis of corrccnuated alveolar macropbagc lysatcs. previ- uusly incubated with I'Illdirsopropyl-plwspfroAurxrdsrtc (DFP), revealed the prescnce of DI7• binding nsatcrial that cornigratod with inactivated HLE. It secros, thcrcforc, that die ckarancc of I ILE by alveolar macrophagcs may represent a significant route fur the removal of this enzyme fnxn the lung. Wlutc, K. Jwwff, A., Gurdurr, R., and Campbell. E. Arntrrcun Review oJResptrarory Diseose 125(6):779-781, 1982. Orlxr srrpporr: National Ikan, Lung and Blood Institute. Frum the Dcputmcnt of PathoWgy, State University ol New York at Stuny Brr><rk. Stony lirout, lhpannrcnt of Pathulogy, Mt. Sinai School of Mcdrc rne, New Yurk; and the fkparuncnt of Mcdretnc, Washmgtun Univcnrty School of Mcdkrnc, St. Louis. 24 25
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SYNTHETI(' ELASTASE INHIBITORS ANI) TIIli1K ROLE IN TIIL-: TREATMENT OF DISEASE Proteases (pnxcin hydrolyzing cnzyn>ts) arc invulvcd in many impunant natural biological prrncsses; they are also thought to be involved tn m:my ddfcrcnt di.cascs. One such discasc is pulnwnary emphysema, which sccrns to result Irunt an uubalancc bctwecn pro(eases n:leascd from human kucocytcs and the protcaxs' inhihxon. Since much cvidcnce has accumulated showing that protcolysis ul' lunti clastin Ic:uls to Ihc dcvclopment ofentphysema, i/ scems cvidcnt that sclecuve cla.uasc inhibiturs could be used in the treatment of tAis disease. The inhibiton used could be natural inhibitors such as a,-prrrtcirwvc inhibitor itself isolated from blood f rx uonatiun, or they could be synthctic matcnals. Work done in the investi);aton' I]INnatUry (U 1)a1C has DhUwn that synthetic elastase inhibitors have considerable p.xcntu) for She trcatnx:nt of cml>hy- sema. Two pcptide chloromcthyl ketone elastase inlubhun Ac-AlrAla-Pru- AIaCH,CI and Suc-Ala-Pro-VaICI(,CI have been shown tu signilicantly duninish thc eatent u(expcrimcntaltlasraxe•itxluced emphyseuw in hamsrers. McO-Suc-Ala•Ala- Pro-Va1CH,C1 has been shown to be orally active in )xuvtdmg protection agatnst induced emphysema in rats. While there arc still qucxions about wlkther such reactive alkylating agents could be used in the lreatment of emphyscma in man, She anunal studies have shown that elastase inhibitors can be used to treat cmphyscma. Tlkrcfurc, based on these studies, it seems that the overall prospects for ,he .kvctupnxnt of a synthetic elastase inhibitor for use in humans in the ncar future uc quite good. Powers, J. C. er u/. (Travis. J.) In: Rich. D. H. and Gross, E. (cds.): PeptiJrs: synihrsis -.+trurrurr - funruun, pru- «r4ings of the seventh American prptir)r symposiarm, Rr>tkford, IL: Pir:rcc Chemical Company. 1981, pp. 391-399. OrAsr srrpport: National Institutes of I lcalth. From She School of Chcmistry. Georgia Institute of Tcchnulogy, Atlanta. PROTEOLYTIC ENZYMES AND THEIR ACTIVE-SITE•SPECIF)C INHIBITORS: ROLE IN THE TREATMENT OF DISEASE Proceases (p(otcin-hydrolyzing cnzyrncs or prwculytic cnzymcs) havc been known fox over 100 years since trypsin was Iirsl isolatcd from panrrcauc )uwce by Kuhne. For most of the period since their discovcry, pr.xcases were Ux,ught ru lx involved only in digestion. However, in dK last dccade, prOxcascs have been shuwn tu be involved in many other important physiulugical Ixrkc»cs, such as lcnduauun, coagulation, arxl the immune response. Outsidc of their m>rnul cnvuunnkut, pru- Icasu can be estrcmdy dcstructive, but natural human plasma inhrbtturs inhthu nrusl prutcases that escape. Irnbalance in pnxease-pruteax inhibitor systcnu can Icad tu a number of diseases of which pulnwnary emphyscma is onc wcll-charait.nzctl csam- pk. Tbis disease resuhs when the protcase clastasc attacks elasun, lin ma)or cla.tii prutun in the lung. Considerabk effort has been rkwxcd tu She syutlx:sis td mhihuurs of pnxeulytic enzymes such as elastase for possible dkra{xutic use. In She luture, specific and selective synthetic proteAse inhibitors should be useful lur uc.,ung.pecrhr dtscases thu range from the common cold to chronic dtwrdcrs such as cmphyscma. Powers. J. C. (Travis, J.) In: Fccnay, R. li. and Whitakcr. J. R. (cds.): Advam rs in ChemistrySrrirs, N,.. 198. A(wGfiratiun of l'rutrins. Washington, D.C.: American Chemical Society. 19112, pp. 3y7•367. OtJursuppurt: National Institutcs of Hcalth. From thc School of Chcmistry. Georgia Institute of Technulogy, Atlanta. SI'E('Il7Cl'fY ANI) RIiACT1VITY OF HUMAN LI.UKOCYTE ELASTASE, PUKCINIi PAN('KEA•IlC ELASTASE, HUMAN GRANULOCYTE CATIIIiPSIN (:, AND II()VINI: I'AN('KI:ATIC CIIYMOTRYI'SIN WI7'11 AKYI.SULFUNYI- 1•I.UOKII)GS: DISCOVI:KY OF A NEW SEKIES OF I'UfENf AND SI'f:CIFIC IRREVERSIBLE I:LASTASE INIIIBITORS The discovery of some prxent and specific inhibitors frx prxcinc pancreatic (P1') elastA.e, human Icukocyte (HL) clastasc, and chymotrypstn Aa is reponed here. In partkular, She reactivity and specificity of a series of substituted benzenesulfunyl Ouaxle with F(Lelastasc, cathepsin G. PP elastase, and bovine chymotrypsin Ao arc des.:nbul. Benzcncsulfonyl fluorides with 2-fluoroacyl substiluenls were found to be potent and specihc inhtbitrxs of clastase. 11L elastase was inhibited most rapidly by 2-(CF,CF,CUNH)-C,H,SO,F. PP clastasc was most rapidly inhibited by 2-(CF,CONH)-C,Ii,SO,F. The 2-(CF,CF,CF,CONH) and 2-(CF,SNH) dr nvatrvcs were quite selective for HL elasusc and inhibited PP elasuse, cathepsin G, and chynxxrypsm Au quite slowly. A specific and potcnt chynxxrypsin inhibitor (2-(Z-Gly-NII)-C.H,SO,F) was also discovered. In this paper, a nwdcl hir Ute clastasc inhibitiun rcaction is proposed which involves interaction of the fluurrrxyl group of the inhibitor with the prrmary substrate recognition site S, of the cniymc. Overall, the results of this study demonstrate that it is practically pu»rhlc to construct simpk organic molecules which ue apecihc inhibitors of HL elastase, PP elastase, or chynxxrypsin. Yoshimura, T. rr al. (Travis, J.) The Journul of Biologicul Chtmistry 257(9):5077-50g4, 1982. FFrom the School uf Chemistry, Georgia Institute of Technulogy. Atlanta. 1{)KMA'1'ION UF A S'1'AlfLfi COMPLEX BETWEEN HUMAN PKUELASTASE 2 AND IIUMAN u,•PKUTEASL• INHIBITOR The studies rcp.xtcd hcrc were auncd at clarifying the nature ul the u,-pratca.c inhibUrx (PI)•b.nrrx) immunrxt:active elastaae 2 in nurrrul plasnn by tnvesttgatrng She intcractnxi uf {u.wclastasc 2 with plasma in vrlra. Results stx)wcd Ihar She ma)ur pro- claslasc 2 binding lactur in human plasma is u,•PI, and Ihat prrxlaaax 2 rcxts dueYQy wilh a,-PI vra a pannl aitrve sne, in a manner sunilar tu that of awve crxlupeptrdascs. Whcn prrklastase 2 was incubated for 16 h at 25'C with 0.1 M dusuprupyl0uoxupMn- phatc, 0.11 nwrl ul rnhtbttix was rncurprx:rtcd/mol of zyrrwgcn. lltc pr.wluct nu longer 26 27
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yielded clastaac 2 following incubuton with bovine ,rypsm. 7lx ditsupropylphus- phuryl-proclauaat 2 was not able to form a complcx with suggesting that the potcntial xtive 3ite scrwc rtstduc in the zytnogen is required (or complcx formatton with a; Pl. Largmao, C., Brodrick, J. W., Gotkas, M. C., Sischu, W. M., and Johnsun. J. 11. Tht lownaf of Biolo jka! Chtntisrry 254(17):8516-8523, 1979. (XJter strpporY: Modical Research Servicc of the Vctcrans Adminisuation. From the Fnzymology Rcsrarch Laboratory, Martinez Vctcrans Admmistratiun McJi- cal Centcr, Mattincz, CA, and the Depuuncnt of Internal Medicine, Untvenrty of Califomia School of Modicioc, Davis. POTENTIAL MECHANISM OF EMPHYSEMA: a,-PROTf:INASfi tNIIIBITOR RECOVERED FROM LUNGS OF CIGAREI'1'E SMOKERS CONTAINS OXIDI7FD METHIONINE AND HAS DECREASED ELASTASE INHIBITORY CAPACM This study was undertakca to test directly whether mcthionine sulfoxide residues could be dcmorutratcd in a; protcinase inhibitor (a; P1) recovered (rum the lungs of cigaruu srubkas. To do this, the clastase inhibitory capacity pcr mg of o; PI was measurcd in the broacboalvcolar lavage (BAL) fluid from 26 healthy smokers anJ 24 nonsmokers. Activity was deaeased by 40% in smokers' BAL fluid compucJ to noosmokers. This cffoct was densonstrabk when either human neutrophil clasux or porcine pancreatic clastase was used as test cnzymc and was rcproducibk when sc- kctod individuals in each group underwent lavagc on rcpeated occasions. In contrast. the fuoctioral activity of a; antichyrrsotrypsin was not decreased in smokers' BAL fluid. Cro.sod aruigcu-rauibody cloctrophotuis conlirmed that inactivation of a,-PI was responsibk for the decrease in the clauau inhibitory capacity of smokers' BAI. auid. Specibcally, a; PI purifud from smokers' IiAL fluids contained methiomne sulfoxide, whereas a; PI from nonsmokers' BAL fluid did not. Smukcrs' a,-PI was indistinguishab(e frorn nonsmokers' a,-Pl on the basis of ekcuophoretic mobtlity, molecular wcight, aad irrurumorcactivity. Thus, oxidation of inethionine restducs in Itrng a; Pl is associatod with cigarette smolting. Bccausc chemical oxidation of ar-PI in vitro causes loss of its clastase inhibitory activity, the obscrvauons prescntcJ hc+c suggest Itut rtsethioainc oaiduioo may also be rclatcJ to dccrcascJ funcuoual acttvity of lung a; PI in wrwkcts in riwr. Carp. H., Mslkr, F.. Hoidal, J. R., arnl Junoff, A. Procttdings of rht Nariono! Acadtmy of Scitncts of rht UnutJ Srults of Ammru 79(6):2041-2045, 19g2. 0rMtrsrlpwt: U.S. Public Hcalth Service. From the Department of Palhology, Sutc University of New Yurk at Siony tlruuk, Stoay Brooit,'atsd thc Depannscm of Internal Medicine, Umversrty of Mtmtesuu Health Sciences Centu, Minucapolis. OXIDATION OF ALPHA,-PROTIiINASE INHIBITOR AS A MAJOR. CONTRIBUTING FACTOR IN THE UEVELOPMI:NT OF PULMONARY fiMl'HYSI:MA t.C3 ~ ~ Thc tmponance of oxidation of alpha,-proteinasc inhibitor (u,-PI) in the Jcvehrp- mcnt of pulnKSnary emphysema is considered cart(ully in this paper. Earlier work has slwwn thal ncutruphil clasuse and cathepsin-G arc respunsibk (ur clastrn dcgradanun in the lung anJ that the majcx funcuon of u,-PI is to contrul elaswaytic activity. Othcr work has shuwn that the reactive site of a,-PI contains a mcthwnyl-scryl pcptidc bond. Trcamcnt with SucNCI chemically oxidizes two of the meMtunyl residues in the inhibitor to the sul(oxidc form, one of which is the reactive site mcthionine. Two ~ methiunyl residues of a,-PI (and no other amino acids) are also oxidrzed by the action ~. of the ncutroplul enzyme, myeloperoxidasc. On NaDudSO; acrylamide gel ekc- troplxxcsis, myclopetoxidasc-trcataf a,-PI has,the same molecular weight as native u,-PI, but canrna (omt a compkx with porcine clastasc. In fact, the molecular weight V of oxuhzul a,-PI is reduced in the presence of elastasc, indicating moJtfication of the inhibitor. Sulucncc analysis demonstrated that protcolytic cleavage occurrcd at the P, mcthionyl burx( and that both the P, and P, mcthionyl residues were oxidized. Also, in an earlier environmental experiment, filxcrcd cigarette smoke, bubbled through plasrna, decreased clastasc inhibitor activity by 3% and untiltcreJ smoke inhibited the activity by 19%. It is krtown, however, that the lungs have a vast surface area, which may rcnder inhibitors on thc lining rrwre susceptible to oxidants. In the data presented in the present paper, an altcrnative process fot the development of a proteinasc- prwcinase inhibitor imbalance in tissues and, in panxulu, in the lung is offered: It is known that many kinds of oxidants arc produced by ncutropltils and macrophagcs. Since a,-PI contains two oxidizable mcthionyl residucs, one essential for activuy, an unbalance between protcinases and protcinase inhibitors could readtly occur. Eventu- ally, such an imbalance could give rise to cmphyscma, even ia inJtvnluals with normal levels of a,-Pl. Travis, J. ct al. Bulletin curoptcn dc Physiopat)wlogic Respaatoire 16 (suppl. ):341-.15I, 198U. Other supporr: Nattunal Institutes of Hcalth. From the Departmcnt of Biochemistry. University of Gcurgia, Athcns. I)lifliRMINAl7ON OF OX1Ul71iD ALPIIA-I-PROI'EINASI:1N11I11ffUR IN S(iRt1M '11tis nictlat<lulugy Papcr Jcsctrtxs an assay for uxuhtcJ alpha-1 prutcrna.w inhtb- itur bascJ un a urcasurabk JrI(crencc bctwcen the inhibitory actrvares of nurm.l and osrJttcrJ u,-1'1 against trypsm and clastasc. Nrxmal a,•PI tnhrbus brwh prrcuk trylnm uMl porcmc parxrcatic elasusc. OxrJueJ u,-PI hu lost rts mhrbn.xy activity toward Ixxcmc pancrcattc elastasc whrk retammg us nct purctnc trypsin inhibitory capxuy, alttwwgh the ratc of associatton with trypsin u nrakcdly rcduccJ. On noting this Jrlfcren.e, a raprd ptucedurc was devclupcJ (or JetcnnrninF ,he pcr- ccntagc of uxidrzcJ a,-PI in plasma or serum baseJ on the ntw of trypsm inhibitory 28 1 29
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t capacity (TIC) tu clasta.u inhibiwry capactity (EIC) ol u,•PI in Utcsc tissucs. Tlx TICI GC ruiu is nut influenced by the otl>tir pnxeins in serum nor by the amccntratrun uf a,- Pl. When this technique was adapted to mcasurc the pr."rnu1n ul' usrdized 01,44 ro U>< serum of young adult, healthy srtwken and nonsmokers. 23% uxrdrzed inhtbitur was found in the urwrkcrs' scra, whereas no oxidized a,-PI was dctcctablc in sera of nonsmoiccrs. Thus smoking rrppeared to lead to oxidation damage to cinulaung u,-PI. Rcductwn of the EIC of the a; Pl in the serum of smokcrs was compensated by a 1.4)- (oid increase in their serum a; PI titers. This assay for oxidizcd a,-PI may bc useful in studies of the rclationship between oxidation of a,-PI and the development of pulmu- rrary emphysenu. lkatty, K., Rubtnic, P., Senior, R. M., and Travis, J. Tlrt Journal of Laboratory and Clinical Medicine IOQ(2):1g6-192. 1982. Udier s.yport: National Institutes of I lealth. IY1Nn the I)elrarWKnl of ItloChenllstly, Unrversity uf Gcurl;ia. AUrcns, an4l 'Ila• Ik•• pxturcnt of Medtcinc, Washington University School ol Medicine at 'Ilx Jcwrsh Ilospital of St. Lwris, St. Luuis. ALPHA,-1'ROTEINASE INHIBITOR IS MORE SENSITIVE TO INACTIVATION BY CIGARETTE SMOKE TtIAN IS LEUKOCYTE t:1.ASTASE In these comparative uudies. aqueous solutions of gas phase crgarcuc smukc were incubatcd with pure human kukocyte clastasc or with crude human kukt>Lyte granuk extract, and thc effects on enzyme activity wcre dctcrmincd using a synthetic amide subsuate. Simultaneously, the same snwke atlutions were incubatcd with 10% human serum undu identical eonditions, and the effccts on scrum inhibition ul purilicd or crude kukocyte clastase werc similarly measured. The in vitro resultd slowed that scrum elastase inhibiting capaacily (a,-PI) is more susccptrble to inactivatron by crga- rettc smoke than is granulocyte elastase, when these proteins arc incubated with walcr- solubk smuke estracts for relatively short times. Similar results were uburncd with both synthetic (amide) and aatural (elauin) substrates, gas phase snwkc and unfrac- tiorwed whole srrwke, and with pure enzymc and crudc Icukrxytc granule extract. Also, aqueous solutions of unfractioruted cigarette smoke were rncubatcd with Icuku- cyte elastase or serum, and the abilities of the smukc-trcatcd enzyme tu dtgcst clastin and of lhe smoke-trpted serum to inhibit elastin drgesuon were dctennined. Both expcrimcnul protocols showed that serum elastasc-mhrbuing capacity (pnmartly caused by a,-PI) is more susceptible to inactivation by ayuerus aulutiuns uf crgarctte smoke Uun is kukocyte elasuse, suggsting that elastasc rdubuion (rather thau clastase activity) may be predomiruntly suppressed by cigarctte snrrkc inhalation in vrvu. JanofJ. A. and l)caring, R. American Raviaw oJRespirutory Disaast 126(4):691•bSN, l5+tf2. Oth.r fr.Oport: U. S. Public lkalth Service. From the Depanmem of P.Uwlogy, State Unrvenrty ul New Yurk at Stuny Bruuk, Stony Brook. 30 GN"I.YMA77C OXI1)ATIUN OF ALPHA- I -PROTEINASE INHIBITOR IN ABNORMAL TISSUE TUNNUVI:R llus review paper describes prcacni knowledge with rcgard to the mtcracutm of alpha•I•fxutcinax inhibiGx (a,-PI) and myclopcroxidasc in vuru, using biah purdtcd cntynk and Iwlymurphonuclcar kukocytes as study souces. In the first place, u,-PI was found tu contain a rncthionyl residue at its reactive center which wa. sensruve to uxidatiun by cuhcr N-chlurusuccinimrdc or by enzymatic oxidation. Chemical uxuta- tiun rcvcated that two of the eight mcthiunyl residues Ixescol in the nwdccuk were oxulrtcd, one of which was at the rcactive center. Subseyuemly, it wa. hwnd that the cnzymatic osidatiun by myclopcroxidasc from ncutrophil granuks, in thc presencc of hydrogen peroxidc and halide ion, could also be easily auained. Sirkc rnyckrpcrox- idaac is present in the samc granules which contain protculyuc enzymes, u would be cxpcctcd that buth types of entymcs would be relcased simultarrcuusly durrng culkr plrag,Kytusn tx ccll tunwwcr. lt is worth noting that the myclulkruxrdax systcm d,rcs mM uxtdiue ra inarlrvare aMrst uther puNCinase inhtbitras ur Itiutcrn.r.cs. In the sccwwl ldxc, rl was slNrwn rcrcnlly that when was cxpuxd to vtahlc neutruf>fuls in the PrescrKe uf ddun,k run and IdxMtarl mynslyl acctatc. Ihc abdtuy of u,•PI to brnd clasta.c was diunuishcd. Iluwcvcr, when mycloperuxrdasc-dchcicnt ceus or tlwse frunr paticnts with chrunrc granulomatuus disease were used, there was no ellcit on a,-PI activiry, tnJrcaung the nwulventent of mycluperoxtdax and hydrogen peruxrde in u,•PI inactivation. More rccently, uxiditcd a,-Pl has been isolated frum rheunatotd synovial fluid of arthrroc paticnts and also from lung lavage flurds ul smoken. Appros- imatcly ftwr mcthiuoyl restducs were found to be uxiditcd in either case. compared to two oxtdizcd residues when native a,•PI was treated wuh either chcmical ur cntym.u- cally paxlur:cd ustdants. The indicatiun is, therefore, that oxidative inactivation ol u,• PI dues occur in vivu. Other studies discussed here deal with the impurtancc of uxidation to cotphyxnw dcve)opment, the artions of the myelupcruxtdau system, and Urc prr»iblc rule of macrophages in the uxidation of a,•PI. Mathcsun, N. R.. Januff, A. and Trur•u, J. Multrtrlar and Cellular Biuchtmutn' 45:65-71. 1982. FFrom the DcpannKnt ut BnKhcruntry. University of (x'urgu. Athens. and the lkpan- nx:nl of Pathology. State University of New York at Stony lSrook, Stony Brook. RA/'ll) CONVERSION OF ANGIU'f ENSIN I TO ANGIUT(:NSIN 11 11Y NIa/7HU1'llIl. ANI) h1AS'1• CI:LL 1'RUTI:INASt:S Aug1lNCnarn II Is a IRINuId that Ir.r.% 1MHCni vasrKrAUtrK9rq arkl d1,141%ICr,nK' x'irC• u.m acuvures arrl rs dcnvcd Irum dre largcr p.rrenl panein angrutauukIl;cn. wMch .ua'ulatcs nt the pl.r>ma. On tlrc uUkr Irand, angiotcnsrn I rs a hlwrdc tlut h.r% no srgnthcanl brulogi.:rl acttvtty arrl requires lunhcrconvcnwn to anl;rutcnstn 11. In,hc stuJres rcpur,cd Iwrc, it is s)Mrwn that human ncutroptul c:rrlw•pam (i and hwuan skin nust cell clrynrax raprdly aurvcn :rngiutcnsin I to angratcn.m II with only nunur ckavagc axwt><rc ra Itx raukcuk lbc nte ulckavagc is.rxuutcnt with a putrnual rulc lur culkr or todh uI these cntymcs in an altcrnatc pahway hu angrurcnsm II 31
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0 synthcsis. Since neithcr enzyme is inlubitcd by captupnl. an angnxcnsin cunvcrttng cnzyme uractivator, it is possibk that kukucytc atxf mast cell cnzymcs may play a signibcant rolc ia the developmcnt of abnorrrully high local conccntr'ations of an- gioteasin 1I. tulsociatod with varwus in0anunaory proccsscs. ReiUy, C. F.. Tewksbury, D. A., Schechter, N. M.. and Travis, J. Thr Jowna/ of BioloticaJ Chcmi.rtry 257(15):8619-8622, 1982. Other sapport: Natiooal lastittrtcs of Hcalth. From the Departmcnt of Biocbemistry. Univcrsity of Georgia. Athcns, the Marshfichf Foundatioo for Medical Resoarch, Marshfield, W I, and the Dcpartmcnt of MeWcinc, Duke Universiry School of Medicine. Durturo. NC. TIlE Ef+IiCT OF a; MACROGLOBULIN ON T111? 1NTliKACI1UN 01: a; PRO"ITINASE INHIBITOR Wfilf PORCINir TRYPSIN 'Rre dissociation of a; prokinase inhibiux:porcinc trypsin cumplcx is rapid and has permitted the expaimcnts reportcd hcre. In this study, the ntc of dissociation of the a; protcinue inlsibitot:porcine trypsin complex by itself was compared with the rate of dissociatioa of the same complex in the presence of a,-macroglobulin. In the prescrsu of the latter inhibitor the dissociation was more rapuf, and active a; pro- tcraasc inhibitor could be rocoversd in the mixture. Huwever, no activc inhibitor could be detected aftu diuociation in the absence of a; macroglubulin. This rccovery of active a,-ptWeirrasc inhibitor from complexes with porcine trypsin is the first demon- stration of a lhcnnodyymic equilibrium between this inhibitor and protctnasc. Cunsc- qucntly, the transfer of trypcia from complexes with a,-proaeinasc inhibitor to a; macroglobulia may be cxplainal as a passive phcnornenon which does not require a physical collision between a; macroglobulin and the a,-protcinase inhibiux:pwcrnc trypsin complex. The dissociation of the complex occurs more rapidly in the presence of a,-rnaavglobulin because this inhibitor complcxes trypsin Icaving the a,-protcmasc idsibitor-.porcinc trypain complcx by both the irrcvcnible breakdown step and by revcrsibk dissociatioo of the compkx. Beuty, K., Tiavis. J. arnd Bcilh, J. Biochunica rr Biophysica Acw 704:221-226, 1982. Other sapport: National lnstitutes of Hcath. From the Department of Biochemistry. Universny of Georgia. Athcns, and the Facu(tt dc pharrrracie. Lnboratoirc d'Enzyrrrologie, Unrventt! Louis Pastcur, Stras(xrurg, France. [:NZYMATIC RI:DUCf10N OF UXIDI7lD a-I-PROfLi1NASL 1N111111TOf( RL'STORES BIOLOGICAL ACTIVITY OxrdatKStr casily inactivatcs a,-protunuc inhibitor thc "taPOr .crum in hibitor of prutcolytic activity. The nutcd inacUvauun sccros tu bc due to the uxnlr 32 uun of an c.sentual nrctluonuic rcsiduc(s) in a,-PI that u rcquircd lur thc inhibition of cla.stasc activity. When rncthioninc ncaiduc(s) in a,-PI arc oxidizcd, they result in Mct(0). In thoory, the presence of an enzyme in cells that can reduce Mct(0) residues in protcins to methiunine provides a rrrechanism for restoring biological acuvity to a protein that has txxn inactivated because of oxidation of an essential methioninc residuc. It has bccn shown elsewhere that extracts of Escherlrhiu rofi contain an enzyme that can reduce Mct(0) rtsiducs in protein to mcthiorrinc. This study shows that oxidized, functionally inactive canine a,-PI does indeed regain its biological inhibitory activity after reduction with a partially punfiod preparation of E. roG Met(0) pcptrde reductase. Abrams, W. R. Wcinbaunr, G., Weissbach, L.. Weissbach, fi., and Brut, N. Proceedings of the National Academy of Sciences of tht United Srarrs of America 78(12):7483-7486, 1981. Other suppws: N+tiunal Ileart, Lung, and Blood Institute. Frum Ure /kpanunuu of Medicinc, Albert Einstcm Mcdical Cenler, Phdadclphu; Ikpartmcnt of Uuklrcmistry and Mokcular Biology, University of h7urida. Gatnes- villc; and Kuchc Instrtutc of Mokcular Biology, Nutky, NJ. NI'_UTROI'I(IL DEGRANULATION IN CADMIUM CHLORIDE-INDUCED ACUTE LUNG 1N1•1.AMMATION Cadmtum chloridc (CdCt,), a toxic chemical that has becn reponcd to cause ccntnlubular or scar cmphysema, was used in this study to induce neuuuphtl migrauurt into the alvcolar spaces as a model of lung inflammation. Results showed that lubar intrabronchial instillation of CACI, (200 µg/m1) in saline causes a reproducible acute pulmonary inflatnmation in dogs. The influx of inflanunatury ncutruphJs from the circulation into thc alvcolar spaces rcaches a maximum appruximatcly 16 huurs after the CdCI, trcatmcnt in thc treated htbe, while the contralateral lung appears rwrmal. Murphumc,rtc quantitation uf pcroxidave-pusuive (azuruphibc) granuks in the inllarm nutory ncurrophils shows a 74% loss of these granules, with little or no krs. nl thc pcroxidasc-negatrvc (specific) granules. These data arc in good agrecnrent witlr the measured Ioss of intracellular elastasc, an cnzymc known to be lucalrzcd in the azuruplulic granules. The results suggest that degranulation of aturuphrlic granuks may occur selectively dunng this chemically-irwfuced acutc inflammaturn. Yanuda, H., Damuno, V. V., Tsang. A-L., Mennze, D. R., Glasguw, J., Abrans, W. R., and WcinAuwm, G. Amtrtcan Journal ujPurh,ilugy 109:145-156, 1982. Other srppoir: National I lcart, l.ung, and Blood Institute. Ftuut,hc Kcxuch Urnst4Nr, Ikpartmcnt uf Mcdtcrnc, Allxrr Eanacm Mcdreal Ccn- tcr, 19rulatklp>aua, ,uul the Franklrn Kcu.uch Centcr, I'Iuladclphu. I'ARAJl'Ml'A'1'IUi 'I'IC'Ml'A'1'IUi'I'IC INNGRVATION OF T11L C1iRV1('AL'fRA('Hk:ALIS MUSCI.L IN LIVING fXX:S (1x uf tt><traihca tu study urw.y luncuun in living Jugs pcrnws csaiutn:rtnm ol a Ixo+rxyptc ccntrai .nrway under rcl.uvcly cunvcmcnt cucunutanres using cstabli.tkd 3-1
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technolugy. In thc study prcxntcd lurc, thc p.rthways by whiclr puasympatlrctic hlw:rs were canied to the cervical tr.cluclis musrle weic .h.nactcntcd in 34 Yncstln:uLCd dogs. Nerves werc Himulatcd ekcuically, and trachcal lcnsnm wu uNrnitoreJ in segments of the pustenor mernbrane in vivo. Somulatrun uf superior larynbeal ncrves contracted 34 of 34 cranial cervical segments and two ul' 1'uur caudal cervical scgnk nts. Rectrrrent laryngeal ncrvcs contributed to inncrvatiun ul' 34 of 34 cranul, u well as four of four eaudal, aegments. Stimulation of paracunent ncrves contracted I I uf 34 cranial and four of four caudal segments. Mechanical cffects of ewphagcal cuntrac- tioo, inducod by stiawlating pararecurnnt nerves, did rwt allcr tension in tracheal segmcnts. Trscheal coatractions induced by stimulation of all three puhways simulu- noously were signi6eantly lr:u than the sum of contractions produced by stimulating ueh se1 individually; this was probably due to anastomoses between terminal ncuruns, overlap in IMir aaatomic distribution, or intercellular ncsuses in trachcalitis muscle. It soems, thcrcfore, that parasympathctic innervation of the canine uaclxa is by thrrc diffcrcnt ncurouratomic pathways. Drown, J. K., Shiclds, R. and Gold. W. M. Journal ofApplied Physiofogy: Respirat, Envirun. 1?acrcuc 1'hysrul. 5)(3):G17-tS25, 1982. OrAsr sryport: National Hcart, Lung and Dkwd Instrtutc. From the Catdiovascular Research Institute and Dcparunent of Mcdrrinc. Univcrsity of California, San Francisco. EFFE(T OF SMOKING A CIGARETTE ON THE DENSITY DEPENDENCE Of; MAXIMAL EXPIRATORY FLOW Earlier studies of these investigators havc shown that tobaccu smuke causcs an increase iaairways resisunce and a drop in expiratory flow at 50%of the vital capacity (FEF.). In the prosent attempt to gain furthcr insight into the naturc and sitc of this brvnctroconstrictive effect, maximal expiratory flow was measurcd in 12 healthy vol- unwcrs while they were breathing air and a low-denaity gas misturc (hclrum-uxygcn) before and aftu smoking a eigarUte. Results showed that prior tu smoking the lurccd vital capacity (FVC) measurod while breaUring air was not significantly different from that obainod whik breathing the heliumoxygen mixture. Smoking did nut cause any changcs in FVC. However, aftcr smoking, thc FEF. mcasurcd whtk breathing air obtaincd dccreascd sigruficantly, whik smoking did not cause any changes in the FEFd after breathing the low-dcnsity gas mtxturc. 09onax„ incrcascd from 47.1= 11.4% bcfort smoking to 57.0 2 13.3% after smoking. Thcrc were nu changcs in flow at 75% FVC and volume of isoflow. These observations arc discussed in light of the cqual prcssurc point (EPP) analysis and wave spccd theory of flow limitation. It was con- cluded rhat after smoking, flow becomes morc density dependent bccarse there is constriction of a flow-linuting segment downstream from the I:PP, krcated in krtrar yal segmental bronchi. No acute effect of tobacco snwke on the small airways could be dcmonsrrucd. Tavciw Da Silva. A. M. and llamosh. P. Respiration 43:258-262, 1982. From the Depanmcnts of Medicine. Physiulugy and lStuphystcs, Gcurgctuwn l)nncr- sity Schools of Medicine and Denlistry. Washington, DC. KI,-SI'IKATORY ANI) ('ARDIOVASC'(1LAR IiFFLCfS OF INTKAVI:NTKI('UI.AK CIIOLLCYSIOKININ In tlus attcmpt to assess the ruk of chulecystokinin- and gastnn-hke pcptrdcs in the ccntral, rcgulatury cuntrol uf respiratory and cardiuvrucular funcuuns. cfrokcystu- kinin in dO»ea uf 1-3Q0 ng was administered inw the lateral brain venutcle uf chlura- losc-ancstlrctizcd cats while tracheal .irfluw, artenal blood pressure, and hcut rate were nwniuxed. Results showe.l that the most striking effect was an increase in respiratory activity. This was observed with a dose as low as I ng arnl peaked with a rkrsc of 100 ng. Respiratory stimulation was indicated by an increase in respiratory minutc volurne, an irkrease that was due to an incrcase in tidal volume as no signrficant effect wu noted on respiratory rate, inspintory time, espiratory tinte, anrl total respi- raarry cycle duratiun. On the other hand, when 300-1000 ng of chrrlccystukinin were administered inuavcnously, no respiratory stimulant effect was ubscrvcd. Thex rc- sulu indicate thal cholecystukinin acts in the brain to stimulate respiration. 1'aKani, F. D., Tavcrra Da Stlva, A. M., llarrwsh, P.. Garvcy, T. Q., 111 and Grlhs, K. A. tarop(an JOMrnaJ of Pharnwcology 78:129-132. 1982. Other support: American I lcart Association. Frum the Dcp:rrtnxnts of Pfwmacolugy. Physiology and Mrdictnc, (ierxgctown Uni- versity Schools uf Medicine and Dentistry, Washington, DC. CIGAKETTE SMOKE CONTAINS ANTICOAGULANTS AGAINST FIBRIN AGGREGATION AND FACTOR Xllla IN PLASMA In this in virra study, the effect of cigarette sawkc on fibrin adgrcKatirn was investigatcd by thc use ol water-solubk, gas-phase components of snxrke, obained by bubbling the srrKrkc produced front one cigarette through thrce ml of bul kr or duulkd water. This cairact was incorprrated in varying dilutions in the buffcr to which hbrin nrunorner w/ution was addcd in order to initiate 6brin aggregation. Results showed a dose-.kpcrukm delay in fibrin aggregation. Iocrcasing the amount of smnke extrxt resulted in decreased absurbance of the clot and delayed onset ol tibrin aggrcgatiorr. The fibnn aggregation inhibitor was also examined by use of two differing tibrin prcparations with u chains lacking COOH-terminal segments. Front the sum ul thcse studies, it was seen that cigarette smoke contains two distmct coagulation rnhibuon: one which prokxrl;s the ckrtting times of plasnu by inducing delayed frbrin aggregatiun and requinng the COOH-terminal region uf frbrin n chains to aren its effect; the whcr inactivates Xllla, thus preventing the cross-linking of fibrin polymers. These antrcoag- ulam prupcrties ol smoke are demunsuabk in plasma, where they may play a ruk in the physiology uf snwrking. Galarukis, D. K.. laurcnt, 1'., Janu,Q A.. and Chung. S. 1. Srrrnrr 217:642-Gi5, 1982. Otlrer sr+Ppart: U. S. Public Hcalth Scrvice. Frum the I lcalth Science Ccntcr. St.rtc Unrvcnity u(Ncw Yurk at Stuny Dru)k, Stony Urouk, awl the National Instnutc uf fkntal Rescarch, Dcttwxla, MD. 34 f 35
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PLATELETS INCREASE NEUTROPHIL ADtIE.RENCE IN VITRO TO NYLON FIBER lntcractwn of neutruphils with platckts is a cuntntun phcnumcnun that may be important in the p.thogenesis of various discascs. Although platclcts arc known to have a strung athnity for foreign surfaces, their effcct on the rncuurcd adhcrcrxc of neutrophi)s in the commonly used nylon fiber systems had not bccn determined. Thercfore, in Ihe study rsrporud hcrc, the cffect of platelets on the adherencc of neutrophils tu nylon fiber was assessed in whole blood s:unpks and purificd ncutruphil wspcnsiuns in the prescnce or abscnce of plasma. Mcasurerncnts showcd that in wholc blood samples, increasing numbers o(platckts werc associatcd with increasing adhcr- tace of ncutrophils. Addition of platelets in plasma to purihcd ncutruphil suspensions increased (p<0.03) ncutrophil adhcrcnce front 76.2''b z 1.4 to 88.0`b = 2.0. Simi- larly, addition of washcd platcicts without plasnu alw increascd (p<0.05) ncutruphil adherence from 67.9% s 5.8 (without added platelets) to 94.2% *_ 1.6 (with 300,(x)0 platekts/rrsm' added). in contrut, no augmcntation of ncutruphil adhcrenic occuncd if platelets had their aggregation response suppressed by prctrcating platclct donors with aspirin. Scanning ckttron microscopy supported these tindings by slwwmg platc- leu in closc association with neutroplrils adhering to nylon fibcr. 'Rk.c lindings ertrphasizc the importance of platelet numbers and reactivhy on the adhcrcnce of acutrophils. Rasp. F. L., Clawson, C. C. and Rrpinr, J. E. The JournaW ojLaborarory and Clinical Mrdicinr 97(6):812-819, 1`J81. OtJursrrpport: American Lung Association, Minnesota Mcdical Foundation. Nauonal Institutcs of Hcalth, American Hcart Association, Knx: Foundation, and the Graduatc School of the Ursivccsity of Minnesota. From the Departments of Medicine and Pcdiatrics, University of Minncx)ta Health Sciences CcrNer, Miwrcapolis, and tAc Webb-Waring Lung Instnute and Dcparfmcnt of lntcmal Modicine (Pulmonary). University of Colorado Health Scicnces Center. Deava. A NOVEL MECHANISM FOR PULMONARY OXYGI:N TOXICITY: PHAGOCYTE MEDIATED LUNG INJURY In initial studics, a significant increase in the numbcr of pulynKxphunuclcar kukocytes (PMN) in the alveolar lavagcs of rats csl>•)sed tu hyperoxia hrr three days was noted. Tlus obscrvation kd to two hypothcxs. The hnt h yputhcsi s, that hyprrux u causes injury to alveolar macrophagcs (AM), inducing their relCalC U( chl11N$dct/C facrors which recruit PMNs to the lung, was suppoxtcd by cndcncc that. (I) hypctuxtA damages AM rn vivo and in vitro, and (2) chcnrtxacuc (acturs ftum alvcular lavages of hypcrusia-cxpuacd animals arc biochcnrically sirnrlar tu tfwx rekaxd by AM cxpuxd to hyperosia in cell culture. Thc second hypothcsis, that PMNs plry an iny+urtam rulc in pulmonary oxygen toaicity, was supported by the finding that the degrcc u(cnrk)4hc- lial damage was highly correlated with the numbcn of PMNs recuvcrcd in alvcul:u lavagcs in borh acutrophrl-wfficicnt and ncuuupcnic rabbtts. Iluwcvcr, twu nulur unknowns still rcnuin. Ftrst, how dkks hypcruxia damage AM7 Sccurx), how du PMNs damage endtwlxhum? At this timc, a rcasunable working hypache.u is that rl>L rlanrage in cach case is due tu the formation uf oxygcn radicals. panicularly thc highly reactive hydruxyl radicals. Improva) undcntanding of the role uf AM, PMN and hydruxyl radtcal in lung damage due to hypcroxia will be tmp.xtant in clucidating basic mochanisms invulvcd in the pathugenesis of pulmonary oxygen tuxtcuy, as well as in other types of envuunmcntal lung injury. Fux. R. lS., Shashy, D. M., )larad'a, R. N., and Rrpinc, J. E. C11t'ST 8()S:3S-4S, 1981. Other support: Anxrican Hcan Association. National Institutcs of Health, and the Mayug-Crawfurd Trust Fund. Frurn the F;xpcrimental Mcdtcinc Division. Webb-Waring Lung Instttute, University of Coluradu Hcalth Sciences Center, Dcnvcr. I'OTf:NT1AL MLCIIANISMS OF LUNG INJURY FROM IIYUROXYL RADICAL Earlier biuclknucal studics havc indicated that inhaled envuunrrrrnul tusins nuy, in one way rK anut)kr, contribute to lung damage. For cxamplc, there is evrdcnce that pulynwrphunuclcar kukocytcs (PMNs) are efficient at nuking highly reactive O; intermcdiatcs and since facton released from alveolar macrophages (AM) exposed tu hypcruxta can stinrulatc release of 0, intermediates from PMN, it appcus likely that 0, intcrnkdiatcs arc involved in this injury. More recent work has focused un the killing of bacteria by PMN as a moJcl of PMN-induccd lung damage. In one of these studia:s, increasing corkcntratiwrs of DMSO or thiiwrea progressively and stgnr ficandy decreased killing uf Sraphylocurrus awrus. 502A, by normal PMN but did n.N further rlccrcase the abnornul bactericidal activity of CGD PMN which tail to paK)ucc •011. Fix atudics with Fe" it was hypothesiud thatS. aurrus was providing a nujutity of thc substu>Lcs(s), such as Fe", which then reacted with H,Q to gcneratc •OH and CH, from UMSO. The results of these studies indicate that hydroxyl radical (•OH) plays an tmpoxtant role in the killing of S. aarrus by human PMN. Other studies suggested that •Ofi might also be formed and participate in the killing uf S. uwrws in anixher way. Elucidation of this latter rnechanism was an outgrowth ol studics d.xtc to dctcnninc the bactericidal activity uf chemical systcm. Fe" and H O:, which gerrer- ates •OH. In the.c studtcs it appears that H,O, reacts with Fe" in the baetcna tu produce •OH by a Fentun type rcacuun. Hydroxyl radical then injures S. uurrus by rcxting with key organic rnulcculcs. It was also rpxcd here that DMSO prevents utjury tu S. uurrus by scavengmg and tktoxifying •OH. Rrprnr, J. E. rt ul. Cf1EJf ri0S:4SS-4riS. 1v81. Ot)Kr support: AnKrKJn IlCart AsslrilatnNl, National Insrnutcs of flcalth, and the Maytag-Crawfurd Trust Fbtrl. FrrNn thc Wcbh-Warmg Lung Institute and the Division uf PulnMmary Mcdw•rrk, Umvcnrty of Cuhuadu Hrilth SctctKCs Center. Denver. 36 1 37
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HYDROGEN PEROXIDE KILL.S STAPHYL(1('O('('US AURF'US BY KL:A('RNG WITII STAPHYLOCOCCAL IRON TO FORM IIYDKOXYL RADICAL In the investigation reportcd here, an attemM was m.dc tu clucidatc the mccha- niscn underlying thc biochcmical rok of iron in bacterial host-rkfcnsc intcractions. Rcsults showed that two diffcrcnl Iines of invcsugatirm supprrrud the prcnusc tlwt killing of Sraphylococcrrs aureus, 502A, by hydrogen pcruxidc involves funuauou uf the rrsorc toxic hydtuayl radical (•OH) through the irun-rkpcndcnt Fenton rcacuon. First, growing S. aa+reos ovcmight in broth mcdia with increasing cunccntrauuns uf ircxt incrcascd thcircontent of iron and dramatically cnhanccd their subxyucnt susrcp- tibility to killing by H,O,. Sccond. in direct relation to their cffcctivcncs.s as •OH scavengers, thiorrrca. dimcthyl thiourea, sodium bcnzrnte, and dimcthyl sul(uxrdc inhibitcd H,O; modiarod killing of S. aureus. Thcrefurc, it seems liont this wurk thrt, whik irorr is an esscruial growth nutricnt, it may alsu pruvide an "Achillcs heel" • for S. aurear, indicating that the reported mcchanism may be a new way in which irun is bcncficial to the host. Repirre, J. E. Fox, R. B. and Bcrgcr, E. M. The Jownul ojBiologira! Chemisrry 256(14):7U94-71Y)6, 19111. OtAer sypport: National Institutcs uf Ikallh, Antcrican Ilcart Assrxwtrun, uNl thc KJCK t•uuMlirutMl. Frwrr the Wcbb-Waring Lung Institute arwl the I'ubrumary Drvutuns ul the Ikprrt- rncnts o( Medicine and Prcdiatrics. University uf Gd4waJu Health Sctcnccs Ccntcr, Dcnver. SERUM FROM PATIENTS WITH INVASIVE FUNGAL INFECTIONS INHIBITS THE ADHERENCE OF POLYMORPHONUCLEAR LEUKOCYT(S AND ALVEOLAR MACROPHAGES Although coruiduabk autcruion has becn directcd recenUy toward dctcrnuning the effect of bactaial infection on ncutrophil adlrcrencc, little attcntion has been paid so far to the influence of fungal infection on phagocytc adhcrencc. In the invcstigatrun reponod hcrc, however, the adherence of various combinations of prrlynwrpMmuclcar kukocytcs (PMN) or alvrwlar macrophages (AM) and scrum from paticnts with fungal infections or from conrru) subjects was evaluated in vrsro using the standard nylon libcr pipette technique. Results showed that the intrinsic arlhcrence of AM and PMN from paticnts with a wide variety of untrcated fungal infections was nurmal, but that these patients had a serum inhibitor that could rcvcrsibly decrease the adhcccncc of PMN arxl AM. Specifrcally, studies using various combinations of PMN and scrums from pa- tieats with blastomyeosis suggested that the adhercnce delcct was due to a serum disorder rather that an intrinsic cellular abnrxnulrty. Whik prctncubauun in scruru from patients with blastornycosis decrcased the adfrcrcrkc of cuntrul PMN, prcirw uh:+- tion in control suum currecacd the dccreased adhcrcnce of PMN Irrrrn patrcnu witfi blastonrycosis. On the basis of thnc anrl other rc latcd studres, rt wa% cuMlu.kd dwt [he intrinsic adherence of PMN anrl AM (rum Paucnts with uutrcatcd fungal mlciuous is normal, but that these paticnts have an catnnstc bcat-I+bile serum Ixtur that can decrease the adhcrcncc of PMN arnf AM. Kasp, F. I... Sarusi• G. A. and Rrp,ne, J. E. Ameri,'un Revrrw rojRe.,prrurory Urseu.re 123:63t`r639, 1981. Orher suppor(: Mmnesraa Medical Fuurxfatiun, MinnesrNa and American Lung Ass. crauuns, Natiunal Institutes of Hcalth, Arnencan Hcart Assuciation, and the Kruc Fuurxfatrun. Frum the lkpartnxnts uf Medicine of the Univenny of Mmncswa Health Sciences ('enter anJ thc Mmncapolu Vcterans Admmistration Mcdreal Ccntcr, and the Wcbb- Wanng Lung Institute and the Departmcnt of Medicine of the Univmuy of Colorado Hcalth Scicrxcs Center, Denver. ANGIOTIiNSIN ('ONVEKTING EN"LYMfi CONCENTRATIONS IN THE l-UNG I.AVAGIi 01; NORMAL RABBITS AND RABBITS TREATED WITL( NITKOGI:N MUSTARD EXPOSED TO HYPEROXIA GranuhKylcs havc bccn mtphcatcd recently in the rkvelupnrcnt u( r'ulc hy- Iw urs tc lung in)ur y, an rdcmatuus lung mislup Ihat is charartenrcd Krusly by nra»rvc avkma and brslulnxWally by crrlod,chal injury with pcnvauular inllantmaurnr. In Ihe lMCM•Irt snN/y, slw•crh.•ally, nKrenKd C1NKentra/Nlnf uf 1mgn7len11n cUnverbng enLyrrK (A('li) wrrc Irwrul in tuug lavagcs (nnn rabbcts expused fur 721nwrs lu hypcrulia and Ihc curKcntratrons uf ACE were currclatcd with ruun of cstravasculu lung watc( to brr,ly wcil;ht and albumut concentrations in lung lavages. In paralkl studtes, rabbits trcatcd with nitrugcn mustard in which granukscytofxnia was maintained throughout the 72-hour hypcwxic esposure period had less cviderrce ol edcmauus lung in)ury and luwcr concentrations of ACE in their lung lavagcs than similarly treated rabbits in which granulocytupcnia was nut maintained. These results suggested that granulucytes contributc (o acute erkmatous lung injury from hypcroxia and that ACE conccntrations in lung lavages reflect this process. Shasby, I). M.. Shasby, S. S:, Bowman, C. M., Fox, R. B., Harada, R. M., Tatc, R. M., and Repinr, J. E. Amerirun Review ojRespiralory Disease 124:202-203, 1981. Orher sapport: National Hcart, Lung and Bloud Institute. Amcrxan Hean Associa- tiun, Natrurwl Instrtutcs of Health, arrl the Kruc Foundatinrt. Front the Webb-Warmg Lung Institute. Department of Medicine (Pulmon.rry Sci- crkcs), Univcnuy of Colorado ikalth Sciences Center, Denvcr. NEU'fRUP1111.5 AND LUNG EDEMA: STATE 01- THE ART lTu hrief rcv,cw Irkuscs un,hc ancrgmg ruk ut ncuturphd..nwl tlw•u pn.duct. rn the dcvclupnKOt tuf adult resprrahxy dutrc» syndunne (ARDS). tlw• uwra aumurm Iwcuntmg Iwm uf crkm.urws lung injury. For sumc rimc• the au.w,stuar 01 ARI)S w,dr nwltrpk miurng cvcnu has led tu tlk prCsunynnNl Ihit, under icnarrr ut:unr ]ta1Kl'\, In.rny IX'trns nuglU hc involved in thc p.lMrgemsu of ARI)S Wlnk rM-l hrurly caabhslw•J, a lus of alvcodar-rapol:uy mcmtxanc macl;rny whwh rr.ults in lung cdcnu appears tu hc t patMdugtc change :rNnrnrm to all cas.s ul ARl).1, thus 38 1 39
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making it possible for cellular or humoral c.>mfxaxnts tu contributc to cndud>clial injury. A nunsber of rcccnt obscrvatiuns suggest that ncutrophds cuntrtbutc lo llw; devck>pa>ent of edcmatous lung injury. First, incrcascJ numbcrs of r>cutruf>l>il. arc commonly found in lung lavagcs of patients with ARDS and early in their dlncss. Soccxrd, neuuophil depiction protects animals from cxpcrin>cntal cdcn>atous lung injury, and third. neutroptuls have bccn shown to have putcnt mechanisms fur causing ussue injury, altering vasculu pernxability and pcnurbrng hcmodynamres. Ovcrall, corrsidcrabk cvidcncc is now availabk to suggest that ncutruphils participatc in the devek+pnstnt of acute edcmatous lung injury such as that seen in patients with AKI)S. The mochanisms by which rscutropfrils might mcdiate thcse cffccts appear to bc many, vrrricd and still ill-dehnod at this time. Rrpina, J. E., Bowman, C. M., and Tate. R. M. CHEST 81S:47S-SOS, 1982. OtRsr support: National lnstitutcs of Ilcalth, Anxrican Hcart Association arxf thc Kroc FourKlation. From the Wcbb-Waring Lung lnstitutc, University of Colurado Hcahh Scicnces Ccn- tcr, Dcnvu. ALVEOLAR MACROPHAGE SECRETIONS: INITIATORS OF INFLAMMATION IN PULMONARY OXYGEN TOXICITY? ., In the paper prcsentcd hcrc, attcmpts were madc lo clucidate nxxchanisn>s respnn- sibk for tscutrvphil influx into the lungs and to undcrstand nwre clearly thc cuntrrbuuun of ncutrophils to the undefrncd paUwgcnesis of pulmonary uxygcn toxicity. For this sWdy, alvcolar rnacrophsgcs (AM). following lavagc frurn lungs of infccuun-frcc Ncw Zealand Whi1c rabbits, were exposed in culture to trornxoxia ( f576 0,) or hypcroxta (95% 0,) for periods up to 72 hrs. Supcrrratuus from AM cultures wcrc cvaluated for their cbemotactic, adhercnce uirnulating, and superoxide radical st>mulating activities for neutrophils. Results showed that hypcroxia damages and stimulates alveolar mac- rvphagcs to rcleasc factors which affect ncutrophil rccruumcnt, adhcrence arul acuva- tion. Prcliminary characterization of these factors suggests that Ihc facturs have separate idcntitics. Factors derived from AM cxposal to hypcruxia differ in molccular wcight, hcat subility, and time of maximal activity. The stimulus for nucrophagc release of these factors rnay involve damage to AM by hyperoxia. These ubscrvations support lhc possibility that AM and the proposed rnechanism of neutrophil rccruiuncnt and activation rruy be important in the pathogcnesis uf uxygen tuxicity anJ other forms of acute lung injury. Harada, R. N., Bowman, C. M.. Fox. R. B., and Rrpinr, J. E. CIIESf 81S:S2S-54S, 1982. Othtr srrpporf: Amuican Lung Association uf Cuhaadu. Anrcn:an Ilcart As.,Kia liun. Nalwnal Institutcsof lkalth, lhe Krtn Frwr>dalruo, 1 WI FuurNlauun, Swan 1'ouu- dal'roa. and Kkbcrg Fuundatron. From the Wcbb-Waring Lung Institutc, Pulnwnary f)ivtswns, Uurvcrsuy uf CuknaJu Ikalth Sciences Ccntu, Denver. OXYGY-N KAI)ICAL-INllUCL•D PULMONARY EDEMA: A MI:CIIANISM FOR THE 1'KODUCTION OF NONCARDIOGENIC PULMONARY l:f)EMA BY NEUTKUPI /ILS Since stimulatcd ncuttuphils makc 0, radicals and sir>ce 0, radicals have been shown to damage a number of biological tissues, it sccmat possible lhu rc kasc of O, radicals from stirrrulatal ncutrophils might perturb the +Ivcolar-capillary rnembnne and lead to protun-rich edcma formatiun. To test this hypothesis, isolated pcrlused lungs from New Zealand White rabbits were monitored and. after a stable baseline pcriod, purinc and santhinc uxidasc were introduced into the pcrfusatc with or without priur introduction uf 0, raJical scavengers. Results of this investigation showed that the chemical gencration of O, radicals by intravascular injection of punnc usrf xanthine uxidasc resulted in acute prwcin-rich cdcrna formation in the isolatcd lungs. Thrs cuknratous process was markedly inhibited by the prior individual injection of several 0, radical scavcngcrs, including catalasc, dirnelhyl wlfoxidc, and dimctf>yphwurca. These Gndings providc direct evidence to wggcst that 0, radicals arc capable of pcnurbing the air-blood barrier and causing a protein-rich edcma. They also suggest that thc intra-vascular release of 0, radicals from stimulated ncutrophils might be capable of producing a similar acute edematous lung injury in certain clinical settings. Tate, R. M., Shasby, D. M., VanBcnthuyscn, K. M., McMunhy, 1. F., and Rrpine, J. E. CHEJT 81S:57S-59S, 1982. Otlur support: Amcrican Lung Association of Colorado, American Hcan A»ucia- tion, National Institutes of Health, the Kroc Four>datioa, Hill Foundation, Swan Folrn- dation, and the Kkbcrg Foundation. From the Wcbb-Waring Lung Inslitule, Pulmonary Division, Cardiovascular PUImo- nary Research Laburatory and the Depannrents of Medicine and Pediatrics, University of Colorado tkalth Scicnccs Ccnter, Dcnver. REDUCTION OF THE EDEMA OF ACUTE HYPEROXIC LUNG INJURY BY GRANULOCYTE DfpLET1ON The relationship between granulocytcs in the lung and the cderna of acute hy- pcruxic lung injury was examined in the study reported here. Results showed that although increased numbers of granulocytcs arc found in lungs acutely injured by hyperoxia, thcir contribution to lung injury remains unknown. It was Gwnd that crrcu- lating granuh>cytes increased markedly in rabbits cxpused to hypcroxra for 7'2 hrs. and that the number of granuh>cytcs in lung lavagcs alsu irscreased and were currelalcd with ttre dcgrcc of crlcruuous lung injury. Funhcnrxtne, when rabbrth wcrc treated with n>trugcn muslard (1.71 ing/kg) and dcvclrK>ed sustaincd gunuGKytupcnra, cxprnure ta hylrrroxia lur 72 lusu resulted in fewer granuknytcs in lung lavaga and less cJcrna- uws lung Injury. In iwurasl, when rabbrts wcre siaulatly ttcated with mttogcn nrustard but did rw>t ur.nnt.nn susu>r>cd granulurytupcnia througl>uul the cxfwsurc lo hypcruxra, u>crcascd nuu>bcrs of granulucytcs were found in lung lavagcs anJ the rkgrcc of e 40 1 41
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alematrxrs lung injury incrcascd to Icvcls not dtffcrcnt Irum those ubscrvcd in oxygcn- expobed rabbits thu bad not been treated with nitrugcn mustard. These findtngs suggcst that granulocyles rnay contribute to production of cdema in acutc oxygen toxtctty. Shasby. D. M., Fox, R. B., Huada, R. N. and Rrpinr. J. E. Journal o/Applicd Physiolojy: Respirlu. Environ. t:xncisr Phy.riuL 52 (5): 1237- 1244, 1982. Orhsr srrpport: National Hcan, Lung and Blood lnstitute, American Hcan Associa- tion, and the Ktuc Foundation. From the Webb-Waring Lung Enstitute, Dcpantnent u( Intcmal Medicine (Puluwnary Sciences Division). University of Colorado Health Scicnccs Center, Dcnvcr. GRANULOCYTES MEDIATE ACUTE EDL•MATOI/S LE/NG INJURY IN RABBITS AND IN ISOLATEI) RABBIT LUNG PEiRFE1Sl-:D WITH I'IIUKESUE. MYRISTATE ACL•TATE: ROLE OF UXYGEiN RAE)ICALS Many funns of acute noncardiugcnic lung edenta arc assx><iateJ with an accunw- lalion of inflamnutory cells in the alveoli and mic:ruvesscls uf the lung, uwl thcrc have boca many suggestions that the activated granulucytes play an intpunant role in this process. In the study reported here explicitly. i1 was seen that intravenously injected phabol myristute acetalc (PMA) caused a prutcin-rich erkma in lungs of control rabbits but rat in granulocytopenic rabbits pretreated with nitrugcn mustard. Spcci- 6cally, control rabbits trcatod with PMA had higher lung weight to body wcight rauus and lung lavage albutnin conccnuatiotts than granulocytopenic rabbits pretrcated with nitrogen roustard and then given PMA. To further clarify the rok of granulocytes in the production of edema, addittonal expcrimcnts were camducted in an isolated pcrfuscd rabbit lung. Additioa of pruificd grarwlocytes and PMA to the balanced salt pcrfusate caused luag edcma, whereas neithcr gramtlocytes nor PMA alone caused eduru. Also, ia order to delcrmiac the contribution of oxygen radicals to the pathogcnesrs of the edema, chronic grauulanuous discasc granulocytes, which arc dcficicnt in oxygen radical produclioa, were added to the isolated lung pcrfusate. Chronic granulumatous disease graarilocytea and PMA did not cause edema in isolated lungs. wlsercas granulu- cytes from rttxmal human subjects and PMA did. Thesc data suggest that oxygen radicals released from uitnulated granulocytes cuntributc to the pathugcncsis of acute aleraatous lung injury. Shasby, D. M., VanBenthuysen, K. M., Tate, K. M., Shasby, S. S.. McMunry, l., and Rcpinr. J. E. American Rtvitw o)<Rtspuuwry Ducuse 125:443-447, 1982. Othsr swppoR: American Heart Assuciation, National Institutcs of Hcath, rrKl the Kroc Foumluwa. Frurn the Webb-Wanng Lung lnsututc and the Cardtuva.cul+r PulnHmary Kr.car:h Lrbcxatory of the University of Colorado Health Sctcnccs Cantcr, fklrvcr. +rkl tilc Pulmonary Divisron of the Uaiversrty of Vitgrnia ScMK>J ul McJt.uk, CharhHtcsvtllc. CY'I'CX'HALASIN U ANl)'fHE: STKU(TUKG OF ACTIN GEt.S 11. FEIRTHE:K EiVI1)EiNCE I-OR TIIG SI'LI7TING U1= F•ACTIN BY CYT(XHAI.ASIN B Cytl>lhalasin li, a fungal tnctabolite that can alter cell shapes and tnhtbit a wide vanety uf ccllular owvcnKnts under ccnain conditions, has been .huwn in the past lu I>L able tu rcducc the network structure of actin filamcnas. In this cuntmunicatiort. alklniunal cwdencc is prescntcd that cykxhalasin 8 sh/atcns actin lilamcnts and that this shluumng takcs place without nct dcpolymerization. Ovcrall, the work dnnc here showed that cyuxhalasin 8 decreased the flow bircfnagcnce and S.. and increased the extinction angle of actin (ilaments in salt solutions favoring pulymcniauon uf the prutcm. These changes occuncd witMwt a detectable increase in ihc cyuthbrium artin niomrmer cunccntratiun determined by a ra/lioassay. Thesc results cumpicmcnt carlier nhscrvauuns indicating that cytochalasin B shonens actin hlamcnts without net de- pldymcnzauon. Analyzcd in tcrnu of Flory's classical nctwork thcory. thr% shortening accounts for thc rnrrkeJ effect of cytuchalasin B in dissolving the gel structure of F- acun crusslinked by achn-binding protcin concentration for incipicnt gclauon. Cyto- chalasm B dccrcaxd the annealing ratc of low concentrations of actm lilarrknt fragmcnts prcparcd by sunic disruption. This result ts consrstent with the idea that cytxnhalasin It Au>,1N tu tl>< ends of actin 6lamcnts, and nuy cxplun how cytuchalasm It causes hl:rutcnt shurtentng. ~ 6laruyama, K.. Ilrrtwig, J. 11. and Smssrf. T. P. lllua himiru ri lliupIrystru Arru 626:494-SW, 1980. Other supporr: U. S. Public Hcalth Scrvice. From the Ucpannknt of Muscle Rcscarch, Boston Biomcdtcal Rescarch Instuutc; l lcmatolugy-Ork/dugy Unit, Massachusetts General Hospiul. and the Depannknt of Medicine, Harvard Medical School, Buston. SfKU(TUKE:OF MACROPHAGE ACTIN-BINDING PROTEIN MOLECULES IN SOLUTION AND INTLRACTING WITH ACTIN FILAMENTS Evidence is presentcd in this paper that acon-bindtng prutc tn is a dtnx r. wht: h has the capacity to inhrrtc and propagatc is.xroprc actin hlanrent networks. For thrs p+nic- ulu study, the structurc of acbn-bmding mukcuks was examined in xdutron rnd /nterxtmg with aitin hlankn/s. At physrologrcal ionic strength, actm-bmJmg paNCtn has a M, value of 540 x 10' as detemined by drrect aad tndircct hydrodyn.rnuc mcasutemcnts. II is .rn assynxtrical din><r compused uf 270 x IU' daltun subunits. Viewed in thc ckctrun nucroscupe after negative starnmg ur low angk sh,nkrwmg. actm-brnding pnrtn tn uwkculcs assume a broad range ut cunlunn:ruuns varying /nxn cluscJ circular strurtures to fully extcmkd stranJs. All cunhgurau.au arc appar.ntly derived fruot the sank slrurture which consists uf twu nwwwxner chains cuntkcted.nd- tu-cnd. Other ldts.mtiuns nutcd in this paper mdreate that actm-tnndrng puMCin duncrs arc caucmcly flexible. In funher studies, direct visualuatrun of arbmbu>,lmg prutcrn nM,kculcs Ititwccn artrn lil:rn><nts in the ckctron mrcrosrlJX .huweJ that Jmn rs ue su/bct.nt ha cro.sbridgtng uf aitrn filanknts and that a.-umhindrng pr/wrm lhmets a1C In/M1IJr, LumpUKll of nNNNnnI'rs lunlKlted hkJ1E-tU-he:Yl JINf having ditrn- m ~ U 42 1 4.1
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binding silcs h><atcd on the (rce tails. Overall, 11 KCnI\ apparl'llt IfU11111K'!l' \1u111c.1 (llat actin-binding protein is a dmrcr at physwluglral uw/c strcngth; each dmur has twu actin filament binding sitcs, and is, thcrefure, sulhclem to gct actin filanients in sulutiun. Thc length and flcxibility of tlu actin-bmding prwein subunits rcrKlcr thls molecule structurally suilcd for thc crosslinking u( largc hchcal tilanrcnts into isutropic netwotks. Hartwig, J. fl. and Srosscl, T. P. Jrwrnal o/MolsculorBiology 145:563-581. 1981. OtAar srrPOort: U. S. Public ifealth Servlcc. Frunl the Dcpartmcnt uf Mcdicurc, Massachuscus Gencral Iluspital, Iiustun. DISTRIBUTION OF ACTIN-BINDING PROTEIN AND MYOSIN IN POLYMORPHONUCLEAR LEUKOCYTES DURING LOCOMOTION AND PHAGOCYTOSIS There is a theury that actin and proteins that asxxiate with it arc the mrjor ekments of locomotion and phagocylusis by pulyuwrphiHwclcar (PMN) Icukrwytcs; and this theory is well suppuned by the nutcrial prescnted hcre. In this study, ilwluccl imnwnufloureseence was used to exanline tlre redistribuuun of myusin alrl acun- binding protein (ABP) molecules in rabbit PMN kukucytes during lucumutiun and phagocytusis. In unpolnrized PMN kukucytes. ABP and nlyosin had a dllluse dutri- bution with some predilection for the ctatcx. In pularized PMN Icukucytcs crawling loward ycast particks, myosin and ABP staining conccntratcd in the antcnur pscudu- pod. In PMN kukocyles frxed during phagocytusis of the yeast partlcks, antimyusm and anti-ABP staining concentrated strikingly in tlk distal ponNms of tlk pxudu(i%,J embracing the yeasa. Staining for catalase, a cytuplasmic prrucln in PMN Icukotytes, for lactofcrrin, a protein of specific granuks, and for myeloperuxrdase, a prutcln uf azyrophilie gnnuks, was rwt eoneentrated in pscudopr>,1s. Takcn tugeQx:r with avall- abk morphologic and biochemical infonnation, these findings arc cunsistcnt wuh a rnechanism wherein intcractions of aclin. ABP and myusin rcdutribute conical cyto- plasm into pscudopods involved in locomotion and phagucytusis. Vakrius, N. H.. Slendahl, 0., Flartwig, J. H. and Stusscl, T. P. Cell 24:195-202, 1981. From the Depanment of Medreine, Harvard Medical School, and the llematulugy- Onculogy Unit, Massachusetts Gencral Hospital, Boston. IDENTIFICATION OF GELSOUN, A CA"-DEPENDENT REGI/LATURY PROTEIN OF ACTIN GEL-SOL TRANSFORMATIUN, AND ITS INTRACELLULAR DISTRIBUTIUN IN A VARIETY OF CELLS AND TISSUES Gclsolin, a 91,U00-daltun gkrtwlar prrxcin frum raldnt Iung IIIJCfU1/11J~1'), IIJ) bcen ahown befort: to be a major Ca"-dcpendcnt rcgulalury pnrrcm ul arun gcbwll translunuatiun. In the work rcfxmed herc, antiscrum prcparcd agalnsl gclsrrlin was used tu dctcct thc prescncc uf pro(cins immunologically rclatcd tu gcl.ulin in a variety of cells usd ri» ucs. Rcsults slwwcd that a single barrd uf cruss-rcaclivc material which cumigratcd with macruphage gelsolin was found in at kast nirre dlffcrent kinds uf cclls and tissues dcrlvcd frum rabbits and humans and in four lines of cultured cclls frrrm humans alxf qts. Gclsuhn was also idcntified in human serum and plasma, raising the pussibility that it may contribute to the clearance of actin from the clrculatory system. Rclatal studics, using indinect immunollourescent staining of acetrxle-tixed macro- phages ux1 polynwrplxmuclear kukocytes, showed that gclsulin resiJcs in the clxti.•al cytuplasm and that during phagucytosis it is concentrated in puudl>{srx/ia engulbng partwks to be ingcstal, an arca of the cytuplasm activcly engaged in movement. In lungitudural cryustat secauns of contracted rabbit skeletal muscle, antlgelwlin staining was asxniated with the I-band of the myofrbril, suggesting that it may be involverf, by an as ycl undehlkd mcchanisln, in skcktal muscle function. In conclusion, Ihc.c lindings arc cumpaGble with the idea that gelwlin is an integral pan of the motile apparatus of phagucytic cclls and that it regulates cell movemcnt by changing tlsc consistency of the cytoplasm. Yin. II. A., Albrecht, J. H. and Fattuum, A. (Stos3el, T. P.) Tht Juurnul ojC'Al Bicdugy 91:9Q1-906, 1981. OOther support: U. S. Public Hcalth Service and the Edwin S. Wcbstcr Foundation. frunr dre I Icrnarulugy-Unculugy lJnit, Massachusetts General Huspltal, and the Dc- pannrcnt of Medicine. Ilarvard Medical ScMtol. Buston. ACTIN FILAMENTS AND SECRETIONS: Tf1E MACROPIIAGE MUDEL In thls (xt-Gllcd brwk chaptcr, the relationship between acun lilarrknts and xcrcuun Is scruunlzcd in several dlffcrcnt ways. In one chapter xctlon, nwrpholugr- cal and pharmaruluglcal cvidence is presented for the participation uf xtin micruWa- ulcnts in sccn:uuu; whlk, in antxhcr scction, a macrophagc model is employed to charactcnze thc denwnstrabk assuilatiun of microlilament rearsangement and secre- tiun. it rs rxxcd here that, if the conical actin microlilament lattice uf rnacrophages rs to have sume actlve ur passivc function in secretiorr, it must be capabk of directional nrwemcnt. Thc ekmcnts of directional nroverrxnt arc (1) a(orce-gcncrating merha- nrsm, (2) an orlcnting inftucncc on the lurce to provide directiunalny, anJ (3) a control mechanrsm caencd on the force-generating system or un directiunabty ur Im both. Each ursc of thcac directional rrxwen>Lnt ekments is prcscnted in fullcr dcuil here. In surrunary, thc lurcc-gencrating mechanism is a superprecipitatiun of aitrn and mytnrn hlaments, a pu><ess reyuinng hydrolysis of ATP and presumably basrd un the sliding- hlament rnteractlun chattenzed in striated muscle. 71us energy-delsendent mechanism may be a uw)ur crxlwmcr uf the macruphage's metabolic activity and can xc.wnr kx tIM 1us1'Cf%If/lhty of xcrcbrnt 161 inhlbitlixl by Inetabul'c purstMs. Du.•clNxMahly arsd amphhiatrun ul tIk tkncc generated arlse fur controlkd fucal dranges in the.russlnrk- /ng uf actln Idanknu. (:clsuhn, a cali lum-acnvaral prutern, contruls lattice rngrduy of I .i.. s;.+ 44 1 45
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+ktin by scvcring actin lilan>Lnts bctwccn Iwtnt. .,l ausshnl ing by xtin-hmJtug prutcin. The frce cakium cunccntrauons that rcgulatc thr xtivny u( this prutcur are kvels found in living cells. This n>Lchanism (or drrcctional cytuplasnuc nxtvcnrcnt control by calciunr is calkd the "Tug-oLWar" hyprnhesis, and cvidcrrca that this mechanism cxists in mrcruphagcs is presented in thts paper. SraJsel, T. P. Mrrhodr in Cdl J7iology 23:215-229, 1981. Other suyport: U.S. Public Huhh Service, the Edwin S. Webster FuunJatiun anJ Edwin W. Hiam. Frum the Henurology-Oncology Unit, Massachusetts Gcncral Huspnal, and the Dc- partmcnt of Medicinc, Harvard Medical Schoo(, Buston. THE MOTOR OF PHAGOCYTIC LEUKOCYTES Thcrc arc three things that are needed for cytuplasmtc muvcmcnt: (I) forcc generation. (2) oricntation of the forcc to provide direction and (.l) a control rnccha- nism. The prescnt rcview summarizes information concerning the biixhcmical compkr ncnts of the motur of rruunrnalian phagocytes and how they can intcract tu gurcntc Jinxtiunal nwvenseM. In the first place, tlre nwtur rcgiun af Itltag0<ylUC Icukucytcs alqrcus tu rcsidc in the pctil>attul c yloplasnt bcncatL the plasma utcudn atK .'f r ansnu.- siua ckctrun micrographs of thin sections of IixcJ phagtKytic kuktxytcs rcvcal th:u this curtical cytoplasm consists prirnarily of hlamcntuus matcnal, anJ that ttrc hla- ments have the dimensions of actin polymers. These actin filaments seem to lurm a meshwork in which thc filamcnts intersect or overlap at random intervals. Myosin mokcu(a (about 1% of Ihe total protcin) of phagocytic Icukucytcs can form bipolar frlaments u physiologic pH and ionic strength, and these myosin filaments bind to actm 6laments. Although all of 1hc evidence is not in yct, at this time it scems pussiblc that: (1) thc macrophage conex is an isotr+opic lattice of actin fdarncnts cross-linkcd by a protein called actin-binding protein. (2) Myosin filamcnts, dispersed together with magrsesium and ATP tAroughout the latticc, exert tension on thc lattice by mcans of the crossbridge mechanism, and (3) depending on the calcium concentration, gelsuhn, a rtccnUy discovered macrroplrage protein, regulates the structure ul'thc lattice. Cumpar- isons are made in this paper betwccn the motor of mammalian phagocytcs anJ the motor of muscle frbers, and consideration is given to the possible imponancc of this motor mechanism in cancer biology. Srosscl, T. P.. Hartwig, J. H. and Yin, H. L. In: Saua3crs, Danicls, Sanw, Roscnfeid, and Denny (cJs,): Fundumrnrul Mrrhu- nismr in Nrurrnn Gracerlrnnrurwlopy. New York: L•:)scvtcr North HullarrJ. Inc.. 198 1. pp. 259-273. OrJrrisrqport: U.S. Public Hcalth Scrvice, the Edwin S. Webster FourrJauun, lalwm W. Hiam, and C. L. and J. D. Kaufman. From the Ilematobgy-Oncology Unit, Massachu..ctts General (luspital, anJ the Ik- partmcnt of Mcdicine, Harvard Ma)ical School. liusrun. 1)li-f1iKMINAN-I-S OF FOKCfa I:XPIKATOKY FLUWS IN NGWf)URN INFAN('S 'fltu study was undertaken to delineate the mcchanical propcnics of the lung in carly life by cxanuning the Jcterminants of forced exptratory flows anJ the response uf flows to HcU, in newborn buys anJ girls. Spocifically, maximal flows at functional residual capacity (Vmas,,,) from partia) cxpiratory flow-volume (PEFV) curvcs (achtevcJ with rapid compression of the chest) were obtained on 11 healthy newborn babies. McanVnux,,, , size corrected by dividing absolute values by measured tbrrcacic gas volume (TGV), was 1.rl0TGVs/s. Specific upstrramcorrluctanccs were high, and the cross-scctional area of the flow-bmiting segment was estimated to be approxi- matcly 0.30 cm' in the throe infants on whom recoil prr:ssures at FRC were also mcasurcd. The cruss-scctional area of the major bronchi in the neonate is approxi- matcly 0.26-0.30 cm'. PEFV curves were convex to Ihe volume axis. Many of the neonates increased their flows while breathing a hclium-oxygcn gas mixture. These results suggest (1) size-comcctod flows ue higher in the rreonalc than in older children or adults; (2) the site of the flow-linuting scgnxnt at FRC during maximal expiratory maneuvers is in large proxirnal airways, similar to the adult; and (3) the relationship of .irway size to parenchymal size may be similar in neonates and adults or, in fact, airways may be larger, relative to parcrkhyma, in neonates. Thercfurc, thcsc physio- logical data do rnx support the hypothcsts, based on pathological studics, that pcnph- ml airways arc Jisproportionatcly smaller (when compared with ceatral airways) in infants than in adults. Tuuaiig, L. M. rt ul. h.unwil 4/ Applrnl 1'hyiudogy: Hri/+uur. t.'nvrrun. h.'srrrur Physiul. S3(S).1220- 1227, 1982. Ollrar srrpport: National Instrtutcs of Health. From the lkpannx:nt of I'cdtatncs, Hadassah Univcrstty Hospital. Mt. Scopus. Jcru- sakm, Israel. CAPI'OPRIL: ASSOCIATION WITH FETAL DEATH AND PULMONARY VASCULAR CHANGES IN THE RABBIT (41446) Captupnl (D-3-mcrcapto-2-mcthylpropanoyl-L-proline) is a rather ncw, orally c(fectivc irrlubnux of angrutcnsin convening cnzymc, which has recently been ap- proved fur the treatment of refractory systemic hypertension. AJmintstratron of this agent has bcen shown experimentally to decrease circulating kvel+ uf angwnensin II and to increase Icvcls of bradykinin and prostaglandias. Fur the study presented here, the cffect of oral captupril on fetal survival was assessed in prcgnam adult New 7aalarwf White rabbits (20 treated with captopnl and 12 controls). Results showed that fctal death tn the treated rabbits was 86`b, in contrast to 1'b in control rabbits. Sonic of Uk rabbits were matk hypuxic in a hypubaric chamber (522 rnm f fg pressure) during tlk pcnuJ of calHUpril admimstration. Under these corsditiuns, captopnl aJmrnrstercJ ttr the rnatemal rabbns had a dcmunstrabk cardtupulrr>,xruy effect in the newrates, as shuwn by a signih.-anl reduction in pulm.uury ancntrlar-mcJta) thickness and b.Mh klr arwl right vcntn.ular wctghts compueJ to the hypuxr.• untreueJ controls. In vrew of these nbs.rvauons, it would be 1>ru.km to avoid using capwprd hu the treatment u/ 46 1 47
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,r hypcncnsion during pregn:uxy, until thc n)cch.nism of fctal rk:ath atxl tlx: rcasrms fax spccics variatioa are krruwn. Kcith, I.M.. Will, J. A. and Wcir, E. K. PrruvrJinas uJ t)u Sor•iery Jor Esprrimtnrul Biology and MrJir inr 170(3):378-383, 1982. National Institutcs of Health. Ot/tersupporti: From the Departments of Veterinary Scicnce, Cullcgc of Agnculuual arrd Life Sciences and Anesthesiology, Medical Schrwl, University ol' Wixunsin, Madison, and the Veterans Administratiun Medical Centcr, Untvcrsity of Mrnncsuta, Minncapolis. STANDARDIZATION OF FORMALDEHYDGINDUCED FI.UOKLSCI•:NCE AND ITS MEASUREMENT TO QUANTIFY SEROTONIN EMISSION IN PULMONARY NEUROENDOCRINE CELLS The furmaldchyde-irxfuced fluorescence (FIF) mcthrx) is widely useJ hx visual- ization and quantitative analysis of biogcnic ammes in tissues. In the paper prescntcd herc, a modifred and surnlardizcd quantitauvc FIF procedure for pnxlu:mg fluorophrxcs and measuring emission intcnsity of xrutonin-cuntaiomg ncuroepuhchal bodies (NEBs) in the rabbit lung is described. This techniquc, using cptfluuresccnic, was reproduced without significant diffcrcnces betwccn control gr.wps. Imfwrtant considcrations frx reproducibility were: using thc samc humidity (1(0')k RII) arxl rcac- lion time (2 hn.) during the vapor uestrnent, sectioning at constant rclativc hwniJity, avoiding unrwcessary heating (sections shoulJ not be strctdreJ over a hut platc) anJ avoiding esposurc of sections to light. Optitnal cnrissiwm readings were ubtained wrrh sectioning rutd mounting at 40-50% RH. Re.rtings wcre reduccJ by 25'%, when the macury light souree was switebeA from 2(X) W to IOU W. 111 was alsu irnprntant to let the instrumcnts warm up long enough to avoid drift during quantitation. Erch Nf'sB should be subjected to the same duration of light exfxnurc for alignment (3O s) befure mcarurriog Quorescenr:e to avoid differences from photodccrxnpusitiun. Keit)r, l. U., Wiley, L. A. and Will, J. A. Natroclunustiy 75:253-258, 1982. Oder srrypori: College of Agriculture and Life Scicnces, Univcrsity of Wrscon.in, Madisort. From Urc Deparuncni of Vctttinuy Science, Univcnity of Wisconsin, MaJtsun. PLASMA PANCREATIC TRYPSINOGENS IN CHRONIC RENAL FAILURE AND AFTER NEPHRECTOMY The study pacsenred here had three majot obIccuves: (a) tu rnvcstig:ne tlx' rclattun betwcea plasma pancr+eatic anionic and cationir trypwxrgcn rn psucnts with chrumc renal failurc or ncpMcctomy, (b) to determine wtktlx:n co:tnuJralysu al(cn tl>< plas1ua kvcb of these zymogcns in paticnts with chronic renal latlurc ((•RF) or nclrhrcctumy, and (c) to study the cffects of nephrectomy on the plasm:r trypsmugcn reslxmsc tu hornwrral uinwlatiun of pancreatic zymogen scuetron m an cstabluhcJ aawtal nxxkl, the Jug. Fur this uuJy, pla.ma cun.cntraaons uf aniumc arxl catiumc trypstnugcn were ux:a.wrcJ in CKf .uxl ancphric paucnts. Kcsults shuwcd that thc plasma cunccmrutons wue signihcantly elevated in both groups of paticnts.. Henwdialysis dkl nut changc their plasnra levels. The plasma levels of anionic arxl cationic trypsinugcns were highly currclatcJ in patients and nornral subjects; howevcr, the relative concentraUUns of anionic trypsinugen were signilicantly higher in renal failure paucnts. This suggcsts dut in patients with renal failure the secondary ekararKx mechanisms for these plasma prwcins clear catiunie molecules more effrciently lMn they clear anionic rrxrkcuks. In a related esperintcnt using normal Jogs, intravenous infusion of synthetic octapeptxk of chok:cystokintn (CCK-8) resulted in small uansiuxy increases in plsma trypsino- gcn kvels. Aftcr ncphrectomy, basal levels of anionic and catiunm trypsinogen were clcvatcd, and intravcrx>us infusion of CCK-8 resulted in prohmgeJ, high kvels of plasma trypsirxrgens. Gtukus, M. C. rr rul. Amerir'an Juurnul of Physiology 242 (Gasuointcst. Liver Physiol. 5):G177-GI82, 1982. Other support: Vetcrans Administration and the National Institutes of Health. Frum Manincz Vctcrans Administration Medical Ccnter, Manmcc, CA; Departments uf Internal Medicine anJ Biological Chemistry. University of Cahfixnia Uaws .1:Mxr1 ul Medtctnc. Davis; anJ the Veterans Administration Wads.vorth McJical Centcr, and Ikpannicnt of Surgcry, UCLA Schrxrl of Medicine. Lcn Angcks. 111. Heari and Circulation SGKOTONIN KGCEf'fOR•MEDIATED STIMULATION OF BOVINF-. SM(>OTH MUSCLE CEI.I. PROSTACYCLIN SYNTHESIS AND ITS MODULATION BY PLATELt:T-DERIVEDGftOWTH FACTOR -fbis papcr reports that scrotunin, an uxlolearnrnc released frum platekt Jcnsc txxlres, stimulatcs prostacychn (PGI,) production by vascular snxxah muuk ccl/. in culture through a spccihc xtutunm-rcccptnr•rrkdutcJ mechanism. It further rcppxts th:rt scrWrmm :rnJ platclct•dcrnvcd growth facwr (Pl)GF) act synergr.ncally to elw'it Jrimauc incrcases m I'(it, synthesin by vaacular smrx)ih mux k.clls ln thc hasr. wurk that led tu thesc cunclustuns. scrolunm (5•hydrusytryptammc: 0 S µM and alxwc) suinulatcJ the synthcsu uf PGI, (as mcasurcJ by raJrounrnunuas.:ry uf h-kctoprus- t.rgl.urdm F,u) by buvmc atxtre snxxnh muxk cells in culturc. Thts dlcct wjs stnw- tuolly spcct6c; a smtdu respimsc was rnN elicited by the otlx:r uxkrks w by ghc ammcs I>rkuylcldrnnc, tsoprutcrcnul, Jopannrrc, or histanunc. llrc resprxtx was rcvcnrbk .i~.. .g..: 48 1 49
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arnd was salurabk at scratumn c.xtcuunuuns of 10 µM or htl;lrcr. An tncrcax in )Yil, syn(hesis was clicited by the additiun of a serutwun agunrst. qutpainu, and rntagu- nizaf by the serotunin receptor blockers cyprulrcptaJnrc. nrcthyscrgtJe, or rncthnxhc- pin. Tbe addition of PDGF to cultures of snwMh muxlc cclls Jramatcally cnhatxcJ PGI, synthesis in rcsponse to the coadministration of scrutunin. PIX:F greatly in- creascd the maximum response to serotunin without altcrtng the half-maxrmal c(Icc- uve conceaturion forsadoain. Also, this synergistic interaction was blockeJ by the addition of a serotonin-receptor blocking agent. Coughlin. S. R.. Moekowitx, M. A., Anwniades. N. N. and Levine, L. ProcttdinFs of the Natiorral Academy of Sciences of rhe United Srates of Americu 7800:7134-7138, 1981. O1As>• sryywR: National lnstitutes of Health. Frum the Departrrsentso(Netrrosurgery and Neurology. Massachusctts Gcncral Hospt- tal, Harvard Medieal School. Boston; Department of Nutrition and Foud Sctencc, Massachusetts Institute of Technology. Cambridgc; Center fur Blo.n1 Kcscarch, Dc- partnr<nt of Nutritioa. Harvard School of Public Ilcaith. Buston, arsd Depannun( of Biuchemistry. Brandeis University. Walt)run, MA. HUMAN PLATELET-DERIVED GROWTH FACTOR STIMULATLS AMINO ACID TRANSPORT AND PROTEIN SYNTHESIS BY HUMAN DIPLOID FIBROBLASTS IN PLASMA-FREE MEDIA There arc two major points about the action of plalckt-dcrivcJ );ruwth lact.a (PfX~.F) on human fibroblasts that cmcrgc frutn this papKr. First, stunulatioHr of mmno acid transpun and 1'li{kucine incorporation are carly ellects of P(X:F action un quiescent human hbroblaus. Second. the actions of PDGF on anunu axid uprake and I'H)kucine incorporation are brought about by PDGF alone and do not rcqutre sitnulta- neous or subsequent presence of plasma componcnts or uthcr honnunes. Spccilica)ly, results of this study show that purified human PDGF induces an incrcase in arnmu acid uptake via system A in quiescent human diploid hbroblasts. Cells must be e tpused to PDGF for 45 rrsin to obtain maximum transport stimulation. Transport stimulation roquircsprotein synthesis; transieru exposrue to PDGF, ahax, in the absence uf plasma contponents can stimulate transport. Acid-insoluble I'Hjkucinc incorporation is alsu stimulaud by PDCF trcatment. and this event also docs nut rcqunc the prescncc of plasma components. Finally. antisaum to PDGF that blucks PDGF-stimul.IcJ DNA syatbcsis in these cells also blocks PDGF-stimulated amino acid uptake arrJ prutctn synthesis. lnerc.ased arrwa acid uptake anJ prutem synthaia tha( occur soon alter addition of fresh suunr to quieseent cells caa be attributeJ to the action u[ 1'llGF acting alone and should be useful as mutcen for the investigation of carly ccllular events eaused by PDCF. Owen, A. J. 111, Gcyu, R. P. and AnronraJas. N. N. Proceedings of rh[ National Academy of Scienc•ts of the United States of Amrru-a 79(10):3203-3207, 1982. Other sopprt: U. S. Public Health Service and the Natwnal CarKCr lnsututc. From thc Department of Nutrition. Harvard School of Public Hcalrh. lirrstun. A NI:W IIt)KM(rNAI. 1'OLl')'fl'Ill)li'111AT S7'IMIILA'II:S'17iG GKOW'I'll OF ('li1.Lti IN ('Ul:fl1KG: ItiO(.AIYON ANI) CHAKACfLRILA IlON I K(1M IIUMAN SI:KIJM AND HUMAN PLA-I'Ia.ETS This prcscntwuun alfurds a wide view of the body of AnuHUaJc% anJ hi% a..uci- atcs. It Jcscribcs brictly (1) their early studies which led them tu the dt.cuvery and ixdauon of a new hurrnunal pulypcptidc growth factorr from human scrurn, wht(h aplxars to bc indispcnsablc for the growth of norma) cells in cul(urc: (2) the subxqucnt isolation and purdication of this pulypepride from clinically outdated human p):rtckb; (3) the growth cffccts• and diverse mel.bulic activilies of this purtlicJ podypcptide in cclls in culture, and (4) studres in pr<7$rt3salmed at thee)ucnfahon of It) rnQde Of al-INHI in thc growth of nurmal cellc in culture. In the work describcd, studies with cell culture and a specific raJiuirnmunoassay demonstrated that the serum pulypcpUde growth factrx dcrives frunr platckts arrd is rekascd into serum during bhnxl clutting; it is mx prescnt in platekt-pocx plasma. This hurrun platekt-dcrived polypcptidc growth factor (PDGF) has been purificd to homogeneity from clinically outdated human platekts. Thc spccitic activity uf thc punhed PDGF is 20 million times greater than that found in unfractiunatcd human serum. It stimulates DNA replication and ccll pruhlcratwrr at a concentratiun of I ng/ml. Characteriiation studies have shown that PDCiF i% a ca- tiunic, heal-stah)c (100°C) po)ypcp(ide, with a pl of about 9.8. The unrcduced nwle- cuk has an appucnt molecular weight of about 35.000 daltons as judgcd by NafArJSO,lpo)yacrylamidc gel ekctruphoresis, and reduction results in the prrxluc- uun of two inactive pu)ypcptidc chains of about 14,ODU and 17,U00 daltons n:spectlvCly. Anruniades• Jl. N. In: Btng, I). H. anJ Kuxnbaum, R. A. (eds.): Plasma and Cellular Alr>.lularwy l'rurrins, Boston: C•cntcr lur BIooKI Rescarch, 1980, pp. 1-14. Other supporf: Natrorul Canccr Instrtutc. Frum the Center (or )(IuuJ Rescarch anJ Harvard University School ul Public Ikahh, Oostun. ))UMAN PLA-I'h1.LT-DGKIVIiDGROWTH FACTOR(PDGF) P(IRIFICATION OF 1')x;F-1 atwl )'IX;F-1) AND SEPARATION OF TIff:IR KEl)UC'kl) SUIIUNfI-S In rhn nxtlNxkdugrcal papcr, a puxedurc that alNiws t)k Ju.•rt rccuvery of biologically acrivc human p)arclct-dcrrvcJ gruwth Ix1u( (PfX;I- );I-) Irunt sumeJ g.)s alrcr scp:rrauun un N.D.~JSO,lpulyxrylamiJt gcl ckarrqrh%xc.u u JcxrtbcJ. Thn tcchntquc crwbkJ the ukntt(icaturn anJ purtficalirn to hnnrugcncily ul' two xtwc 1'IX;F polypcptiuks, urn with a nMrkcular wctght of ahrwt.15,(Xx) (PI)GF-1) arrJ the oUwr with a nwlccular wctgh(uf ab.wt .1?,00U (PDGF-11)• RedurcJ PIXi)=-l prrrJutcJ two rnacUvc subunits, with rrrukcular weights of about IS,(X)0 arw) IS,ODU. KrJuitJ P)X;F-)I alsu pruJurcJ twa rnarttvc subuniu• with rnukculrr weights uf abuut 1 S,OUO and 16,(XX). )t is I>u» tbk: that 1'IX;F-1) derives Irunt prutculytic ckavagc of PD(iF-I. ~ ~ +^~ 50 1 51
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Aruun+uJrs, U.N. YrurvrJings of rhe National Ar•w/rmy of Srirnrrs of the UrurrJ Jrurrs u/ Amrriru 7g(12):7314-7)17, 1981. OOther support: National Institutcs of Hcalth. From thc Ccntcr frx Blood Rescarch and Dcparlmcnt of Nutnuun. llarvard SChtN)l ol Public Hcalth, Boston. STIMULATION OF PIIOSPHOLIPID AND CIIOLtSTI:R()l. f:.til'l:R SYNTHESIS BY PLATELET-DERIVED GROWTH FAC"iOR IN NORMAL AND FiOMOZYC:oUS FAMILIAL HYPfiRCHOLIiSTLROLEMIA HUMAN SKIN FIBROBLASTS Thc aim of this investigatiun was to study thc effcct uf human platclct-d.:nvcd growth factor (PDGF) on lipid nxtabolism, particularly cholesterol cstcr and phospho- lipid synt)sesis in nornsal -and familial hypcrcholcsterolcrnic-dcnvcd human skin fibroblasts. Results of this study show that purc PD(:F at narwgram kvcls sumulatcs cholesterol cster, phospholipid and DNA synthesis in both normal and f:amilial hypcr- chokstcrokmia mutant human skin fibroblasts. Whrk sumulatwn uf DNA.ynthc.i. did not begin until 15-24 hrs. aftcr addition of PDGF to quiexent normal and FH mut.nt fibrobfasts, stimulation of I'Hiokic acid incurporation into chulesterul cstcr and phospholipid was evident 3-6 hrs. aftcr the addition of PDGF. In the nonnal ccll., thc rate of choks{erol ester synthesis was maximal at 24 hrs., then rapidly dcclincd. CTolesterol cstenficatioa was much bwcr in the FH cells thut in the nrxrnal cells. The stimulation of I'Hjokic acid incorporation into clwkstcrul cstcr by 1'IX;F was mbtb• itul in both rrumal and Fli mutant skin lrbroblasts by Ixugcstcronc, ao mhrbrux of acyl-CoA: choksterol acylvansferase. llrc rate of clwlestcrul estcr syodw.u in the normal cells increased as the concentration uf platckt-ptwr plasma ur low denstty lipopraein (LDL) was iacrcasc.d, especially in the presence of PIXiF. Liucaricaurm uf the LDL dose-respoase curve indicated that PDGF increased the rate rathcr than the affinuy of the ovuaB choksterol esterification system. The rate of cholcsterul esten- hcation in the FH mutant cells was highest in the absence of LDL or at low kvels of platekt-poor plasma. Consequently. PDGF can samulate cholesterol cstcr synthesrs by LDL and non-1.DL-mediatcd processes. Lcslie, C. C., Anwniadas, N. N. and Gcycr, R. P. Bioclumica rr Biophysica Acta 711:290-304. 19g2. From the Department of Nutrition, Harvard School of Wbltc Hcalth, Bustun. MIGRATION OF CULTURED VASCULAR CEI.IS IN Rf:SI'ONSIi TU PLASMA AND PLATEI-LT-DERIVED FACTORS in the study presente.f hcre, a yuantitativc assay Irx cell mrgratuun was u.cd to mcasure the response of pencytes, xmxHh muxle ccll% and crxkrthclwl cell. Irrrn' large and umall vessels to human serum, plasma and purtlkd platckt-denvcd lat,turs. In tlk hra part uf tlus study, phagukincuc mrgratmn uf culturcd vaKUlar c.Ils was tested rn resprmse tu human platclet-rich scrum ('scrum'1 and human platckbpuur plasma serum 1'pla.nu'). The cell types lestcd included bovine aurtK erwkNhchal cells, hunun umbdical vcin cndrNhchal Cclls, human hcmangrsxnal capillary cr><knhe- Iral cells, bovine adrenal microvascular pcricytes, arxf bovinc aurtk smooth ntusck cells. Hutuan scrum stimulatcd a srgntficant increase in the rate of mignttun in all live cell types. Huruan plasma stimulated the cndolhclia) cells to mtgratc but had no effect un the mrbra/iun of pencytcs or srnorxh muscle cclls. In the second part of thu study, highly puntied platclct-derivu) growth factor (PDGF) stimulated dux-dcpcndcnt mr gratrun of snrwth nruaclc cells causing a S0`b increase in p)ragukmctk track arca relative to controls. Nenhcr pencytc nor crK3uthelial cell migraurm was .umulated by PIX:F. Rabbit auttscrum to human PDGF cumpktcly blocked UK snt<x,th muscle cell migratiun induced by either 10% serum of Ing/ml purc PDGF. Purilicd platckt factor 1 V(PF,) stiutulatcd migration of pcncytes but not uf snworh muscle cclb or cndrxhehal cells. Sheep uuucrum to human PF, completely blocked the pcncyte mtgratiun rn- duccd by either 10% scrum or I ug/ml purc PF,. These results indicate that PDGF is the pnnrary factor in scrum responsible for the migration of culturcd aonic smrxah muack cells and that PF, is a cnucal factor required to induce the migratirm of pcncytes. Other factors present in brxh plasma and serum control the migrauon of vascular crwiuthclul cclls. Bcrnstcin, L. R., AnronruJrs. H. and Zcttcr, B. R. ' Journal ujCrll Srirnc•r 56:71-82, 1982. OOther support: National Institutcs of )kalth. From the Dcparturcnts of Physiology and Surgery. Harvard Medical SrMwd. Chil- Jrcob Medical Center, and Iluvard School of lbblrc Health, Ccnter lur Bluud Re- starch, Boston. 1'l.A'I'I;LLT-Ut:RIVED (:RUWf•H FACTOR BINDS SPECIFICALI-Y TO Rl'.Clsf'fORS ON VASCULAR SMOOTH MUSCLE CELLS AND TI1E BINDING BECOMES NUNUISSOCIABLE 1'latekt-Jcnved growth factor (PDGF) is a putent stimulant uf thc gruwth of vax ular smooth musc{e. In order to understand the action ul PDGF, mettwrJa had w be developed for idcntrfying PDGF receplrx sites. To accomplish thrs, radrurudtnated 1'IXiF (I'll-PU(iI-) was used in studics of PDGF binding sitcs on vascular snwuth musclc cells. llkre was an cxccllcnt currelation between the ability ul "'1-PUGF tu strnwlatc cell pruldcraOrm ar><1 to bind specdically tu cultured vaatulu .nwuth muxle ccll. Thc hall'-nuxmraJ cunccntraurm hx both processes was 0 I nM There wcrc 5l),lXri) buxlmg suc. per ccll. Reduced PDGF, prepared by tseatnknt ul P1X:F with 20 mM dnhanhrenul. had ncither the .bihty to band to snrouvh muscle cells rxx to snnm- late cellular pnthlcratiun. lipnk:nrul growth factor, ncivc growth fartrx, hbrublast growth faaur, anJ huturk B did mH cumpetc fur the binding srtes at a cunccntrauun uf 10 nM. "1-PUGF binding waa slowly revcrsibk at 4'C and was rapidly and IrNally ~ ~..,.~ w T 52 1 53
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nversibk aftcr a I-min incubatiun at 3TC. Altcr cuuunucJ inruhauun at 37'C. t1K binding bccunc irreveoibk. The haU-tinre for fontW run n/ t1n nunJus"xtrhlc statc uf '^)-PDGF was - 5 min at 3TC. Thc noodissuciabk statc ul binJtng was not GxurcJ at 4'C even after I hr of incubation. Thcse dau suggcst that the IahclcJ sttes arc the PDGF rcccptoa thau rrsediate PDGF's mltllgcnic action and that a M)nd1%MKlahk .llJlc of PDGF binding is forrned at 3TC. It is Iikely that nunJisxxiablc PIX:F rclxC.cnts intcrrralizcJ ligand or binding to sita thal arc convcncd tu a higha(limly statc a(tcr thc ligand binds. Williams. L. T., Tremble. P. and Antonfodes, Jl. N. Proctrdings of she Natioaol Acodcmy of Scicncts of the UnirrJ Staras uf Amtrtru 79:5867-5870,1982. OtJrrr strppat: National lnstitutes of Health and the Mihun Fund of HarvarJ Mcdical Scbool. From the Cardiac Unit, Massachusetts General Huspital, Ik»tun, anJ the Center for Blood Researeh and Deputtnerrt of Nutrition, HarvuJ Univcnity, School ol Public Nca)th. Boston. CONTRIBUTION OF PLASMA PROTEASE INHIBITORS TO THE INACTIVATION OF KALLIKREIN IN PLASMA This study was designed to assess the rcspcctive contribution uf the Jd(crcnt plasma protcasc inhibitors to the inactivation of kalhkrcin in plasma. The use of a new technique that sekctively inactivatcs a,M in plasma (acilnateJ the cumparisrm of t)x: kinctics of kallikrein irracuvation. Resulu showed that. under pscudu-lirst-utJcr cun- ditions, thc inactivatiou rstc constant of kallikmin in rsunnal plasma was 0.60 mut'. This ruc constant was rtduccd to 0.35, 0.30, and 0.116 min', in plasma Jehctcnt respectively in Cl-INIf, a,M or buth inhibuuns llws CI-INII (42%) and u,M (5(Nk.) wcrc found to be the major inhibitors of kalliktcin in nonnal plasma. Murcuvcr, all thc other protease inhibitors present in normal plasnu contributed orrly for g% to the inactivation of the enzyme. To conbrm these kinetic results,'°I-Kalhkrcin ( M, 85,(X)0) was compktely inactivated by various plasma saoiples, and the resulting mtstures werc ana)yzed by gel filtration on Sepharose 611 CL for the appearance uf "'1-kalb- krein-inhibitoreornplexa. Ovuall, the results presented hcre tndicate that Cl-INI I and a,M arc the major inhibitors of kalhkrcin in normal human plasma. This conclusion is supported by (a) the analysis of the kinetics of kalltkrcin inacuvatiun in nurnwl and pt<stease iahibiwr-deficieut plasny, and (b) by the yuanutatron upon gcl hltruion of the kallikteia-inhibitat eompleses fonrred in plasma as the result of the inactivation of purihcd radiolabckd enzyme. Schapira, M.. Scott. C. F. and Culmun, R. W. lowrnul of Clinical Jnvcstigarion 69:462-468, 1y2t2. Or1Kr suppaY: National Institutcs of tkalth. From the Thrombosis Rescarch Centcr, Temple University School u( MeJicnte. Phrladclphia. 111(;11 MOLI:('()1.AR WEIGIf(' KININ(X;FN OR ITS I.IGHTCHAIN I'ROTIi('CS IIIIMAN PLASMA KALLIKREIN FROM INACTIVATION BY PLASMA I'RO'I*UASH IN111BITORS t:.2 ~ ~ Ftvc plasuu prutcasc inhtbrtrxs are known to inactivate kalbkrcin, but rw:r it has p..( bccn shuwn al.u that high molecular weight kininogcn (IIMWK) ur its light chain can act to Ixutect kalfikrein from inactivation by these inhibttora. For this biochemical study, tlrc kmcucs of the inactivation of kallikrein by a,-macrvglobuhn, antit)vonrbin Ill, and a,-antrtrypsin were nuted in the presence and abscnce of HMWK. In the abxrrce of IIMWK, the second-order rate constants t.,tK, for the inactivation were rc.pcctivcly 6.9 x IIY. 1.8 x)0', and 2.5 x 10'M 'min '. When HMWK was present Junng the reaction, the inactivation ratts by thcse plasma proteasc inhibitors ~ were reduced as a result of the fortrwion o( kallikrein-high molecular weight kininogeo .~ contpkx, K, = 0.75 µM. When the light chain derived from reduction of kinin-free ~ IIMWK was substitutcd for the parent mukcuk, a more pronounced reduction in f.-. inactivation racs was ubaerved due to thc formation uf s kalhkrein-light chain cumpks (~ (K, = 0.14 µM). Thesc results dcmunstrate that the combining site for kalltkrun utr IIM WK, rcspomsibk for complex formation and protection agarnst tnhtbiturs, resides in the light-chain region of the nwiccuk. Mrxrover, kalbkrein appears to bind mure tightly to the Itght chain of HMWK than tu the parent molecule. Schapira, M., Scott, C. F., James, A., Silver. L. D., Krrcppers, F.. James, H. L.. and Culman, R. W. Bior•hemistry 21(3):567-572, 1982. Other support: National Institutes of Hcalth. Frum the Thrumbusrs Research Center and the Department of McJicine, Temple University Hcalth Sciences Center, Philadelphia. RI:GIONAL KGNAI. ANf) SPI.ANCHNIC BLOOD FLOWS DURING NICOTINI: INF•USION: EFFECTS OF ALPNA AND OF COMBINED ALPflA AND BETA ADRL:NI:RGIC BLOCKADE This study was instituted frx the purpose of defining directly thc roks of ulpha adrcnergte arxl Ixi»ibk nunadrenergic mechanranu in vaso:,mstnctur resp.mxs tu mcwinc. Tu du thu, nicwine-induced changes in blood Aow and vaxularconJuctarrcc in regional renal and splanchnic beds were evaluatcd before and afrer (1) sckcttvc blockade of ulphu adrenergic receptun and (2) conrbirrcd blockade uf ulplru and /vm adrencrgic rcccpurrs. l)efurc adrenergic bluckaJe, nicotine incrcascJ artcrial pressurc (+82'b) but haf heterogcncuus directional effects rwr regirxul blood tluws: puwreas (-(4'1k). dwalcnum (- 33%), kidney concx ( - 31~ip), kidney ok-Julla 1- I7'.{ ), liver (+ 596) and spkcn / f 7196). Vaxular conductancc was rcduicJ in kidncy crutr)t, kidney mcdulla. Jw><lcnum, liver and pancrcu, and was rwt altcrcJ ur spkcn. Scka uve alpha aJrencrgic bluckaxk prevented the hypertensive respunsr to ntcrNrne, but hctcrugcneuu. changes in regional Quws persisted. Alter cumhrneJ ulphu .nd hrtu ' :rJrcncrgic bhKk,wk:, mcottnc tncrcaacd systemrc ancnal pre»ura arx) rkcreaxJ 54 1 5S
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vascular corw)uctance in all tissues. Results indicate that tlrcre arc (1) a hctcrugcrrcuus in/iucncc of nicotine in renal anJ splancturic crrcularuns assuciaaf with rcgiunal differences in activities of alpha at>,l Dew adrcnergtc receptors and (2) a potent rrrn- adrcncrgic vasr>Lonstrictor response in these circulatwns to nicutiue alter bhx:kadc of alphu anJ bea adrenugie receptors. Downty, 11. F.. Crystal, G. J. and Bashour, F. A. ThtlournaJ of PlwrwracoJogy and Exptrimtnral Thtrapturucs 22(k2):375-381, 1982. OtIisr s.yport: Cardiology Fund. From the Deputments of Physiology and Inlcrnal Medicine, University of Tesas Ikalth Scicnce Centerand Cardiovascular Rcscarch Laburatury at Methodist Huspital, Da)las. EFFECT OF CIGARETTE SMOKING ON HIGH DENSITY LIPOPROTEIN PHOSPHOUPIDS This uudy was designed to assess the effect of acute inhalation of agarcue smuke on high density lipoprotein (HDL) phospfwlipid compositiun. In running thc tcsts on White Carneau pigeons, the following four treatmcnt regimens werc instituted: (I) shelf control birds fed a chow diet and rctaineJ in thcir cages; (2) sham pigcons fcd a choksterol-saturate4 (u rliet and exposed to fresh air by a smoking machine; (3) low nicotine-low carbon monoxide (LoLo) animals also (ed the choksterol-fat diet anJ cxposed to low cwrcenuatioos of thcsc cigarette sawhc products; and (4) high nicut mc- high carbun monoxide (HiHi) binls fed the cholcstcrol-fat diet and subjected to high cuncentratioru of these inhalants. Results of these studies showed that the chukstcrul drer caused an increase in the concentration u(most HDL phuspholipid classes. lspu- surc to the HiHi regirnca resulted in an increase in the I Il)L chulcstcnrl/f>lwsp(rulipiJ ratio amf a reduction in Iht: concentratiorr of IIDIL plxtsphatidyl elhatHdamine, f>1Nrs- pharidyl scriaelinositol, splringornyelin and lysuphusphattdyl clNrbne. It appcar~, therefore, that cigarotte smoking may attentrale IIDL's anti-alhcrugcnic properties by altcriog surface pbospfqGpid components. Hegany, K. M., Trugiu, L. E., Mulligan, J. J., Cluctte, J. E.. Kew, R. K., Stack, D. J., and Hoirwcki, J. L. BiocJumical and 8iophysicol Research Communicarions l04( I):212-219, 1982. Ot/ssr srrppat: American Heart Association, Greatcr Boston Division. Fran the Dcpartment of Biological Sciences, University of Luwcll, Luwcll, MA. ALTERNATIVE COMPLEMENT PATHWAY-DEPL•NI)IiNT lN(ifSTION OF FLUOUTE PARTICLES BY HUMAN GRANULOCYTES Partrcksof a fluort:seent cyclic hydrocarbon (Fluuhtc) have hecn rcpuncJ bclore to be ingestui by rnonuauekar phagocytes of hurnan blood. Thu paper describes quantdativc stuJic, ul the mgcstiun ol Qrcx same panidcs by hunun granulocytcs. In thc stuJics prcx:rucJ lurc, f-)uufitc p+uticlcs with an avcragc sve uf l) lµM were ingcstcJ try hunran granulr>Lytcs ahcr rncubatiun in fresh nunnal hwnan scrum. Inges- 1rrNt, which was assx:sxJ by visual counring in a ffuuresceM microx'upe ul cells crNnaintng particles. requueJ fresh nutmal serum arwf did not occur whcn scrum was IKatul fur 30 minutcs at SU'C or when ethyknuliaminctetraacetic acid (EDTA) was present. Particurarly, it did not occur in serum genetically deficicnt in C3b inacuvatur ur in C3. Huwevcr, phagocytic activity was restored to C3-de(x'ient serum by purified hunrur C3 and to hcar inactivated serum by purifial(actur B. Opsonrzatiun of particles undcr appropriate conditions is insensitive to the absence of human C2 or C5, but is rkpcndcnt upon C3 and an intact alternative C pathway. Measurcmcnt of the opsonir,- hun of these particles thus constitutes a simpk assay of the functional integrity of this pathway. Although the mechanism involved here is still unckar, this simple and readily availabk assay can be used clinically as a screen fur the functional opsunk actrvity uf thc alternative C pathway, as well as for the capacity of human granulucytes to recognize and ingest C3-coated particles. Arnaout, M. A., Luscinskas, F. W., Uonani, F. J.. Alpu, C. A., and Valcri, C. R. Thr Journal ojlmmunology 127(1):278-281, 1981. OlJur srrppoR: Ofhce of Naval Research. Frrmr the Division u( Ccll Biology anJ Ncphrology. Children's Hu.pital Medical Center, Boston; Centcr fur Blood Research, Boston; and Naval Blood Research Labo- ratury, Buston. I:NI)OTH(:LIAL M1: fABULISM 'llk:rc arc thrcc tnq>.ntant lurxuons u(cnJudxlrum-t I) sclurauon uf bhrMf anJ lymph rrom the canvascular space, (2) contrul of the inllux anJ clllux ol spccilk bhKxl wlutcs anJ cullords, and (3) pruvisrun of a sm.wth gliding suAarc tur passtng bluthl-that have been wcll recognized lrw years. The buuk chapter prcxnted here. tlwwgh, deals mN with these three functions but with the metabolic acttvities of the crxkwhchum. While crKhxhclial cells arc well-equippcJ for glycolysis. axidauvc phur pMxylatiun arwl the complex series of reactiuns required for cell divtsam, it is the number of far-rcarhimg metabolic activitics, nwst Jiscuvcrcd since 1968. that arc cunsirlercJ here. Tu wit. crwAnhclial cells pussess a highly crxnpkx array of rnuarcllu- Iu nurhiucry anJ can, tn fact, elafxuate nwkcuks and macrunMrkculks un{>,wt:rnt rnN rrnly lu cell uucgrity url cell division but also rmpxtant in tcnn. u( prrw'c»mg vasuactrve and hcnwstau. subatances. Many uf the recently rccugnvcJ nwtabolic xtivnics occur at ur ncar the cell surfarc. Fur exanrpk, angtdcn.rn-iunvcn+ng cn- rynk yrp.:an tu tw dispuscd su that tIK cnzymc rtxlf is cmtarkkJ in the plasnu nrcmbrarx, yet ns catalyuc site is srtuatcJ to have accc» to anghNcn.m I arw) braJy- kmm as tlky pas tn circulating blood. One section ul this paper Jcals with ;uuhNm:a) iomsrJcr.wun.. ainl arr.xhcr with survcying,h. cnduUkhwu's knuwn uw tabuth' artrvr- tk.. While nwch uf thc wurk urr cnrhnikbal nkuMtlunr lxrhKrrxJ.u Lu ha% b..n arcwnplultcJ thruugh the Jraciplures of bsochcmutry. murplxthogy. :rnJ cell bnrl.rgy. 56 1 57
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uther studics, physiukr[tic, ph unwculugtc, arld pJIINI{or li, arl- Il':LIIbIG, and IhCre 1i every rcawn tu belicve that clinically rcl.:vant J.rta will be luntxunung. Ryan. J. W. and Ryan. U. S. In: Wicdman. M. P. (ed.):An lnrrwJut•riun ofMic•rorrrruluuun, New York: Acadcuuc Press, Inc. 1981. pp. 147-169. Orka supporY: U.S. Public Health Service and the John A. I lartfurd Fuundauun, luc. From thc Dcpartment of Mcdicinc. University of Miami School of Mcdicine. Miami. FL. STRUCTURAL BASES FOR METABOLIC ACTIVITY The pulmonary endothe)ium, which is the hrst linc of cwttact between bkwd• brxne substrates and the cellular nuchincry of the lungs, is rcvicwed here along with those uructural specializations that are suited for their role in the metabolic functions of the lungs. The underlying structural bases for metabolic activity arc crxtsickrcd here undu the Uuoe headings: anatomical, eellular, and subcellular. Particular emphasis is placed upon the enzytnes, inhibiturs, receptors, and transport mechanisms that are important in the regulation of the hormonal composition and fluidity of blood. Fulktw- ing anatomical atudy, a picture emerges of a varicty of cnzymcs that arc situated on thc surface of pulmonary ersdodsclial cells and arc strategically poised for interacuun with the appropriate wbetrates delivered by the blood and arc equally strategtcally placed to ddenoinc the quarYities of active substances allowed to pass downstream or into the extravaseular space. Cellular studies show that the cavcuiae-plasma membrane (rac- tioa of lung bomogenate eonverts angiutensin I to angiwcnsin II and degrades brady- kinio to yield the charaneris(ic products formed by intact lungs. FurthcrnKxe, the preparatron can degrade ATP and S'-AMP to adenosinc and frce phosphate. By use ul' a sensitive radioassay, it was alw shown that pulmonary crKknhclial cells contain a[wn- daN angiutensin converting enzyme (ACE). In sununary, it appears that Urc uniyue features of pulmonary enzymes may rclatc to thc structure of the lungs and to the positioa of the lung in the circulatory system. Thus, the twal anrwnt of ACE within the lungs may greatly eaceed the amount needed to pntcess the cuncentrauun of angiutcn- sin I and bradykiaia usually found in pulmonary artcry bkwd. Whik it is known that ACEoccurs on the luminal surface of these cells, u ts becoming evident at a suh:cllular level that the nxrnbrane bound enzyme is scnsitivc to (actun such as uxygen crNxcn- tration. However, the effects o(components of the plasma membrane on the nwlecular coafiguration and bence on the activity o( the enzynsc arc nut known as yct. Ryan, U. S. Annwo( Review oJPhysiuJagy44:223-23`1, 19112. Otha syppv.t: National Institutes of Hcalth. Frum the Department of Mulicine. University of Miauu Schuul of Mcdreme. Miami, FL. NI'.UTROPHILS ARE RI?QUIRI:D FOR THE DNA SYNTHIiTIC RESPONSE OF II[1MAN 1.YMa'i/OCYTGS TO MI:VALONIC ACID: IiVIDENCF: SU(:(;I:ST1N(i'1'1fAT A NONSTEROL PRODUCT OF MEVALONATE IS INVOLVED T/k ability nf varnuus enanuonxnc forms Of inevatunic acid to initiate lympho- cytc DNA syntlksa is cxamtned in this paper, and the ruk of the ncutruphdic puly- nMnl>awrnuclcar IcukrKyte as the helper cell in evoking nkvakuwlc-irrluccd IymplKx ytc prulderauun is defined with greater precisiun than has bcen accompluhed bcfurc. The ability of lymphocytes to initiate DNA synthesis and cell cycling is a radioscnsitivc pnq.erty of the cclls, whcreas the help pnuvidcd by ncutruphils is nnin- taincd despite their priur cxposurc to x-irradiation. Other organic aCid anwns, includ- ing prccursor% of mcvalunic acid biosynthesis and a variety of products of mcvalunate mctabulism, fail to initiatc DNA synthesis when added to human lymphocytcs. Be- cause only thc metabolically active R( -) enanliumcr of inevalunic acid inniatcs lymphr><ytc DNA synthcsis, it seems probable that physiological pathways of rnevalo- natc maabolism arc involved. The response to rncvalonic acid of ML-236B (compac- tin)-inhibitcd lymphncytes is incrcascd, and the threshoklconcentraarxr of mcvakxutc at which lymphtxyte DNA synthesis first sppears is decreased, when the cells are cultured in lipuprutein-containing medium. The response to mcvalunic acid of lym- ptxxytcs cultured in hpuprutcin-dcpkted rncdtum can be enhanced by adduiun to the cultures of low density lipoprotcin but rat by addition of high density lipaprotcm. Based upon the flux diversion hypothesis of rnevalonate metabulism, these ubscrva- ti.ms suggest that a nunsterol product of inevalunatc metabolism may be responsrbk fur the initiation of lymphocyte DNA synthesis by mevalonic acid. Larxrn, R. A., Chung. 1., Scanu, A. M., and YacHnin, S. Prw•teJings r J the Nuric.nu! Academy of Sciencta of the United Srurrs of America 79:3021/-3032, 1982. Other suppoit: U.S. Public Hcalth Service and the Naku Center Research Fund. Frorn the Ucpartmcnb u( Medtcrne and Biochemistry. the Franklrn MrLean Memorial Research Institute, and the Committee on Inununology, The University of Chicago Schuul of Mcdicinc, Chicago. MEVALONIC ACID IN CONIUNCTION WITH HELP FROM NI:UTROPHILS INDUCtS DNA SYNTHESIS AND CELL CYCLING IN HUMAN PERIPI I[:RAL BLOOD LYMPIIOCYTES Mcvalonic a.id plays an unprxtant rule in tax rcgulauun uf nsunnulr.tn cell growth uKl drvunm. Results from earlier studies have even sugEeslcd a cntic.J role Irx nrcvalumc arid, tndcpcndcnt of itu conversion to chrdestcrul, in the regulation ul DNA synthcsis and cell rcplicatnrn. Evidence presented in this paper sAuws that nkvakrnic -wid does stimulatc DNA syntlksrs in hwuan peripheral bluud lymphrwytes whrch have been iwl:ucd by gravity xdrn>totNirm of blood and frced of adlkr.nt ccll. by nylon crJuton passage Ilunun pcriplkral blood urunwsurkar cclls i.ulat.d by the Fkrdl- Ilypayuc tcchnryuc rca>.url lcss wcll. but their re.punx to m.val.wui acid can he cnh,u><cd by the rrutaphd-rkh Frcull-Ilypayuc-iwlatcd "bwtum" acll Irarulin. The klnetlCs Uf mlvakNnl acNI-UNIucCd IyrllphWytC tran]IonININMI are 1rn11ldr tU thU1l' Ul nKrrc classri lymldxcytc nutugcns. In :xk/rnun tu sumul.rung lymphu:yt. DNA syn- thcsu. ukvalumr xkJ pn,,duces a population uf ccli. rcprcsrntrng alI PhJ.cs ut Ilr cell 58 1 59
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I cyclc whose nwxplxrlugical charactcruhcs arc typIra l uf thu.c .ccn wilh nrure runvru- tiunal milugcns. The DNA synthetic respunx uf lymphuryt.s tu okvalunic actd cau be abolished by priurcapuaure of the lymphocytes tu x-irtadFauour ur tnhuwnycin C, while the helper ef(ect uf granulocyles is unal'fected by cither ttcautknl. These obscrvaliuns suggest 1ha1 mevalunie acid mN only nray play a rule as a critical suhstaia'c whit'h wpports Urc prupugation of cells prugrammcd tu diviJk, ur stirnulatcd lu drvidc by various initiNors of cc11 growth, but alw that, in su.ccptiblc ccli h.qtulauuns, nneva• Ionic ac4f may act as an inJrrccr of tlsc cntirc program uf nce ccll cyclc. Yachru'n. S. and Richrnan, D. P. Cellular lmmunology 72:24g-262, 19g2. Orbersr.pport: U.S. Public lieallh Service, Muscular Dystruphy A»ociauun, and tlic Nako Rcscarch Foundation. From tlre Department of Medicine And Neurology. the FranAhn McL-can Mcnwnal Research Institutes, and the Commiuec on Immunology, The Univcnhy of Chicago School of Medicine. Chicago. IV. Neuropharmocology and Physiology NICOTINE BINDING SITES AND THEIR LOCALIZATION IN TIIL: CENTRAL NERVOUS SYSTEM The study of nicotine binding in Ihc brain, which was plagued with prublcros during earlier investigatiuns, has bcen helped greatly by thc dcvch>pn>Lnt of a nxalnMl of obtaining the unnuurai (+)-isuatcr optically pure and by lln: refined synthcsis of radiolabekd nicotine with high s)scci/ic activity. The actions uf the stcreauomcn of nicoline on the central nervous system are qualitatively similar in utust tcsts but (-)• nicwinc is nrrxe potent Uwr the unnatural (+ )-isuuicr by at Icasl IU-fuld. BrrK)int; uf radio)abckd nicotine to brain has both salurabk and nonsaturabk conrypuncnts. Only saturable binding is affected by incubation conditions such as time, temperature, pil and ion conceatration. Excess eoncentratiuns of tln stereuisuroen are equally cf Iccti ve in displacing (-)-('HJ-nicotinc from brain hurnugcnatcs. Ncvcnhcless, a direct cum- parisoa of (+)-1'H J-nicotine and (-)-1'H J-nicotine binding shows that the latter has a K, three times luwer than the former. (-)-1'H J-nicutinc is bound to the grcatcst degree in hyputhalamus and hippocampus, areas that also exhibu the most slercusckctivity liw nicotine. However, differences in the binding aftiniNes of the two isxarrers are far Icss than the pharmacological stercospccifrcity observed. MaAin, B. R. and Accro. M. D. Neuroscierrce 6c 8ehovioral Reviews 3(4):473-478, 19>S 1. Other sappoa•t: Nationa) Lnstitutes uf Health. Fnrm the Department of Pharmacology, Medical CoUcgc uf Vtrginia, Kidunurrl. (rU ('l1AKACPGKI'l.ATION OF NICOTINE BINDIN(i IN MOUSE BRAIN AND 1' COMPARISON WITH THE BINDING OF u-BUNGAKOTOXI": AND ~ QUINUCLIDINYL BE:N'LILATE: ~' T The litenuuc suggests that nicuunc binds to ncurun:d lisNue. but Ate nature And sigmficaiKe ul tbn buwhng arc in question. In thn study ol mcutine bindtng, the burdin); uf 1'11 Jmrutiuc lu nKw.e Main was nrcasured And subsequently cumpared with O tlk hirMhng uf 1"'IJu•bungatutuain (u-BTX) and L-J'HJ yuinuchdmyl bcnztlatc F (QNB). The bux)ing uf nicotinc was saturable, rcvenibk and stcreospccdic. The (, average K,, and B_ were 59 nM and gg fmoks/mg o( protein. respectrvely. Although the rates of assoKiation and dissociation of nicotine were tempcrature-depcncknt, the ~ incubation tempenture had no effect on either K„ or li_ When measured at 20'or 3T, ni:dinc appeared to bind to a single class of binding sites, but a sceond, very low- afhnity, binding site was observed at 4'. Nicotine binding was unaffected by the addition of NaCI, KCI, CaCI„ or MgSO, to the incubation medium. NicWtne chu- linergic agunists were putent inhibitors of nicotine binding; huwcver, nicutim: antagu- nists wcre poor inhibitors. The regional distribution of binding was not uniform: mtdbrain and striatum contained the highest number of recepton, whereas cerebellum had the fcwesl. When nicotine- a-BTX, and QNB binding were compared in several ways, results indicated that all three ligands label cholincrgic sFtcs, but these shcsdiffer from onc another. Differences in site dcnsities, regional disuibutKrn, inhibitor potcn- cics, and thcrnul dcnaturation indicated that nicotine binding was not the same as chhcr QN B or u-BTX binding, and therefore that «ceptors for nicoNnc may represent a unique population of cholincrgic receptors. Marks. M. J. and Collins. A. C. Mukcwlur PJwrmurolul;y 22:554-564, 1982. Other support: National Institutes of Health. From the Institute for Behavioral Genetics and School of Pharmacy. University of Colorado, Boulder. 1iFl-L('I'S OF ACUTIi CGNTHAL. AND PERIPHERAL ADMINISTRATION OF NICOTINE ON I IYI'O17IALAMIC CATECHOLAMINE: NkRVE TERMINAL SYSTEMS ANl) ON THIi SECRETION OF ADENOHYPOPIiYS):AL HORMONES IN THG MALE RAT The actiuns of intraventricular tnjcctiuns And tntravenous mluauns of mcuturc wcrc cvaluatcd un dupanune and noradrerulme stores and turnover tn dt.crch' hyixrha- larrric dupanurrc And nuradrenahnc nerve terminal systcros in nulc Spraguc-Dawky rats. Antcrxx prtunary hurrrwnc secrctiun was alw studied in the same group of amnuls. Spccthcally, mcasurcmcnts wcrc madc uf LH, FSH. GI1. TSH, prulactin and crxbcoacrunC scrunr kvels ustng radwimmurwlugtcal p(uccJures. lntranrhvrlual arnclauoms bawcan regional cateclNrlamine kvels and tumuvcr and htrcmorrc s"re- lUUn wcrc pcrl.irnkd. Kcsul4 showed that intraventricular in)ativun% ul m:utmc pra- du.•cd Jusc-d.pcnrknt reductions uf dupamine and nor.drcnahnc kvcls And tncrra.cs uf dupanuru And txxadrenaline lumuver in ducrete hyputhalarnrc arca.. The lurnrvcr 61 .~. Z. r10V t;.~.
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increascs were assuciated with reducuons of arruut Icvcb ul' TSfi, prul.rcun and 1.11 and an incrcasc of serum FSH kvcls. Currclataun analy.u in tlk mtravcntricuh,r cxpcrimcnts was perfurnred. Alw. intravenous inlustuns of ntcutinc over unc hour pnx(uccd duse-deperrdent raluction uf dopaminc and nuradrenaltnc cuncentrauuns and incrcascd t)re turnover of these monoamines in the vuwus hy(xnhalamic carcchu- lamine nerve ternrinal systems analyttd, with the nwdian cnuncncc, dupammc aud nrsradrenahne nerve tcrnrinal systcnrs showing thc higl><at senauvity to nicounc. Overall, the total of these and other cxpcrtmenul lirrdings reinforces Uie view thrt nicotine nray act dirmrly on the brain to activatc dupaminc and nuradrenalrnc nerve ternurul systcros, probably via activatiun of nicotine-likc chultncrgic rcccpturs. C'urrc- lation analysis shows Ihu other ncurolnnsmittcr nxchanums must alw be involved in producing the nicotinc-induca(changes in the secrctwn of antcrror pituitary hunnuncs. Anr(crsson, K., Fuxe, K.. Eneroth, P., and Agnab, L. F. Medica! Biology 60:98-111, 1982. Other suppori: Svcnska Tubaks AB, Stuckholm. From the Department of Histolugy. Karulinaka Inatitutct, and thc I lurmunc Latrna- tory, Kuvlinska Hospiul. Stockholm: and the IXpanmcnt ol Fiuman Phyaiulugy, Uaivcrsity of Motkna, Modena, Italy. ON THE INTERACTION BETWELN NICOTIN(i ANI) CYCLOIIEXIMIDI: Inhibitors uf protein synthesis, such as cychnccxtmidc. havc been shuwn tu a(lcct merrrory rctcntion and nicotine has been shown tu revcnc ttre arnnc.uc prupcnacs uf cyclohcximide. In the study presented here, the imeractiona of nrcuune and cyclulk u- imide on brain protein syMhesis were examtncd in an attempt tu Jetcnninc whether the nrcwine-induced reversal of the amncstic effect of cycluhcsuni.k u due tu an tmcra.•- tion berwecn nicotine and cyclohcxrmidc on brain prutem aynthcsis, and whether n involves the sites in brain that saturably bind I'H jnicotinc. Rcsults ahuwed that nrcwinc did rsol revene the cyclotteximidc-indtrced inhibition of protein synQuan, both rn vivu (inun animal) and in vitro (brain slices), suggesting that on-guing protcin synthcsia n not necessarily involved in memory consolidation. Alw, thc nkuune bnMhng aitca were nut affected by in vivo or in vitro treatment with r:ychdreximidc citlrcr in ttk presence or aLscnce of nicotinc. Scrshca, H.. Reith, M. E. A. and LajrJw. A. Brain ResearcA 251:183-185, 1982. From the Center for Neurochemistry, Rockland Rcscarch Inuttutc, Ward'a Ialand, NY. EVIDENCE FOR A NONCHOLINERGIC NICO77NG K(iCL•'PTOK ON IIUMAN PifAGOCYTIC LEUKOCYTES Radioligand binding studres show that IcuktK ytcs cunta1n ., numtx•r ul {x pu& and hormorrc reccptors, such as thosc for chcnxxactuc pcpttdcs and .r.ctylchuluk. In Ihis I I attcurpt tu Icat tlw hypudxsts that nicotinc acts aa a rcceptur (known ur unknuwn), the apeca/ic binding uf 'l-I-(d,l) mcotinc to Icuk(nytcs waa mcawred. Kcsults uf thu study denMmstratc the prcaence of a nonchulincrgic mcutirrc reccptrx on human phagncync kukucytra. Thc average afhni/y + / - standard deviation of (d.l)-nrcwinc for the rcccptor on ncuwrophils is 36 + / - 18 nM (n=6). The binding u saturabk with an average of 8.7 x I lY sucs Ix r neutruphil. Monocytcs atr), to a kucr extcnt, lympho<y- Ics but 004 crythrucytcs alw display specific binding. Bound nicotine is di»ociabk (rum tlre rcccptor .uxl is nut mctabuhtal. Only close stnrctural analugs uf nrcottnc bind (u drt rcceplur, which ts stcreusckctivc lor the (d)-iwmcr. The rcceptor can be occu- pied by (1)-nicuune at cuncentrations prescnt in the blood of smokers. It is suggested tlrat wntc of the advcrsc ef fccts of snrohing on kukocyte functions may be mediated by a specific nicotine receptor. Davtcs, B. D., Hoss. W., Lin, 1-P., and Liorrclti, F. Molecular and C'rllu/ar Buxhrmistry 44:23-31, 1982. Orhtrsupport: U. S. Public lkalth Service. Frunt Qic Ccntcr lor ISrain Kcscarch, University of Ruchcstcr School of Medicine and fknustry, Rochcstcr. NY, and Center for Blood Rcsearch, Boston. CIIAKAC fERI-l.ATION OF THE ISOLATED PERFUSED MOUSE BRAIN AS A SYSTEM FOR NLUROCIiEMICAL STUDIES The critcria lirr characterizatiorr of isolated pcrfuaed brain preparauons mclude the rknxmstrsuun of ekctrical, structural and metabolic viability. 'flre purpose of this study wa.% to meet these critcria and to cstablish the isolated pcrfuscd moux brain (IPMB) as a viable rnrx(ci for ncurochemical and neurupharmacolugical atudica. In the paper presented hcrc, the preparation of the IPMB rs rlcxribcd along with ita ckc- trophysiolugical, nxuplxdogical, biochemical and pharmacological propcnres. Using high performance liquid chromatugraphy with clcctrochcmtcal rktecuun, the primary mctatsuhtc of nrammalian central nervous system norepincplvinc, 3-mcthoxy-4-hy- druxyp(rencthytcncglycul (MIiPG), was measured in the pcrfusane at 1S-min uucr- vals. The rate of MIiPG prodtrction was similar to literature values of the rate of rrxcpurephrinc turnover in mouse brain. MHPG pruducuun rate in the IPMB was bkKked by pretrcatnrent with 6-hydruxydupamrne and was increased by pretteatrncnt wtth reser)ntk. Tuwcll, 1. F. and 1:'rwtn. V. G. Brarn Rtsrwch 209:476-481, 1981. Other support: U. S. Pubhc 1lulth Service and thc Univcrahy of Colorado National Ccntcr for Akulrul Rcacarch. Frum the School of Phannacy, University of Cokxado, Boulder. ANAI.YSIS OF REGIONAL VARIATIONS IN TIU: AFFINITI(:S OF MUSC'AKINIC AGONISTS IN THE RAT BRAIN The spccalii binding uf radiolabelcd antagonrstb has been used tu charxtcni.c the muxannrc acctylchulrnc reccptur in bratn. Fur the study reported Ircrc, thc aftinrues u/ 62 i 63
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muscannic agents in brains from male Spraguc-Dawky rats were rktcmuncd by direct and indircct assays of binding to the receptor. Rcsults sMrwcd that the brain stcm of the rat has a higher aftinity toward muscarinic agonists than dkKS the /ixebrain. Receptor occupancy eurvu of both rcgions of the brain deviate Irum simple nuss-acuun bind- ing. 7Te characteristics of the binding in each region are compatible with the exutcnce of two non-interacting binding sites, and arc not attributable to Jcscnsiu[atwn or to ncgatively couperative binding within a snull oligumcr; howcvcr, the possibiluy of large oligomers remains to be cxcluded. The agomsl binding data werc analyzed by a linear transfonruuion of Seatchard-like inhibition curves of the binding u/' thc antago- nist I'H/yuinuclidinyl benzilate. Such analysis, based on uMxkl of two subpopulations of rceeptors in each arca, shows the subpopulalions of the brain stcm and the forebrarn to be distinct. Brain stem: 44% of receptors pussess high alliniry with dissociation constant for carbachol, K, - 2.8 x 10-' M, dissoctation constant of luw-afhnity nxcptor, K, - 2.3 x 10 • M; forcbnin: 41 % high aflinity, K, = 2.1 s IU ' M. K, = 1.7 x 10'' M. The data suggest that whole brain contains at least threc maJur muxannic receptors which can be diuinguishal on the basis of their aflinrtics for agonists. Ellis. J. and /luss. W. Brain Rexarch 193:189-198, 1980. OrBer sryport: National Institutes of Health. From the Center for Brain Research, University of Rochcstcr School of Medicine and Dauistry, Rochcstcr, NY. COMPETITIVE INTERACTION OF GALLAMINE W1TH MULTIPLE MUSCARINIC RECEPTORS Neural membranes from the brains of male Sprague-Dawky rats were prcparcd and used for binding studies with gallamine and carbachol. In the paper prcacntcd hcrc, it is shown that gallamine, a nicotinic antagonist with antimuxarinic potency in xvcral systerns, interacts competitively with the tritiated ligand /'111yumuclidinyl bcnttlatc (QNB) at the muscariaie rcceptor. The occupancy curves derived from these studies suggesl that gallamine has widely varying affinities fot dtllcrcnt subpupulatiuns of rnuscarinic ncceptors, a finding which sets gallaminc apart frum classical nwsrarinic anugonists such as atropine and QNB. The grcatcst difference in a(/inttics lur Kal- lamine uccurre.d in the brain stem, whcrc the data could bc satulxtunly liued tu a twu- site rrr>,1cl, with 77% of the rcccpuxs having hrgh afbmty (K, - 25 uM) arwl 23'X% luw affinity (93 p.M). Further, these affinities displayed rank order cunelatmg with ttwnc 4,1 carbachol (an agonist), although gallarninc has not, so far, duplaycd agurust (or partial agonist) activity. The finding that antagonists as wcll as agunists can display multiple afhnities for muscarinic receptors suggests that there arc fundamcotal dillcrcnccs anang subpupulatiwu of these recepwrs. Ellis. J. and Hoss. W. ' Biu.•Irrnural Pharmacology 31(5):873-876, 1982. Orhsr s+rpporr: National Instituwcs of Health. From the Center for Brain Research, Univershy of Ruchester Schwl ol Mcdreutc and Dentistry. Rochestcr, NY. i V. Pharmacology and 13iochemislry AN ISOLATEf) PI:KFUS(iD DOG-LUNG PREPARATION FOR THIi STUDY OF CYCLIC GMP MEiAI)OL1SM: EFFECTS OF SODIUM NITROPRUSSIDE AND OXYGI•:N In recent years, considerable imerest has developcd in the rule that cyclrc GMP may play in pulmunary furxaion, partly because the lung has a high guanylate cyclasc acuvity compared with uthcr tissues and paniy because the lung is in direct contact with vanous cnvuonnxntal pollutants, many of which arc known to precipitate the fonna- uon of oxygen free radicals and danuge the lung. For the study prcsented here, the intact, isolatcd pcrfuscd dug lung was evaluated as a modcl for studies directed at dcfining the role of uxidalivc modulauon of lung cyclic GMP mctabuGsm in pulnrunary functirxt. Sidium niaoprusside added to the pcrfusion blood increased the cyclic GMP content of lung over four-fold in a duse-dependent manner. Although wdrum ndru- prusside administrauon cauxd changes in lung vascular resistance, these occurred indc{xndcntly of the changes in cyclic GMP. Ventilation of lungs with a high oxygen gas mixture containing 95% 0,. 5% CO, acutely increased the cyclic GMP contcnt of lungs after IS minutcs from 1.3 ±0.06 to 3.4 s0.12 pmol cyclic GMP/mg protein. Cyclic GMP levels returned toward control during continued vemiluion with the hrgh oxygen concentration. The uxygen-irsduced ekvatioa of lung cyclic GMP content was nut accompanied by changes in lung vascular rcsisWtce. The results indicate that the isulucal perfused lung may be useful in studies of cyclic GMP, tissue oxidation and pulmonary function. Bruwghltr, J. U. and Maron, M. B. t'wropran Journal ojPharnwcology 78:187-193, 1982. Orktr supporr: Arncrican Lung Association. Frum the Programs in Phannaculogy and Physiology. Northeastern Ohio Universtties College of Medicine. Routstown. INVOI.VI:MIiNT OF SI)LFIfYURYL GROUI'S IN TUE OXIDATIVfi MOI)ULAI'ION OF 1'ARTICULATE LUNG GUANYLATE CYCLASE BY NITRIC OXIDE AND N-MfiTIiYL-N-NITRO-NITROSO(:UANIUINE This reprut rlcacnbes the activation of paniculate rat lung guanvlate cyclax by nunc oxide and N-orcthyl-N-nitro-N-nitrrrsuguanidrne (MNNG) and suggests the in- vulvement of sulfhydryl groups in the activation process. In thu study, paniculate guanylatc cyclasc from rat lung was activated by nitric oxtdc tw MNNG in a dusc- tkpcndcnt mannrr that was affected by dithwtMenul. AltMwgh low cowtcentralnms of mtnc oxide or MNNG pnrluced near nsaximunr activuiun, excessive autuunts de- crcaud the particulate cnryme activity. Nitric uxidc-uimulated guanylate cyclasc x•tivny decayed during a 6U-m;nute preincubauon period at 37'C, but did not decay at 24' ur 4•. Dnhiothrcitul cnhanred the decay ef nitnc uxide-stimulatcd enzymc at all tcmpcr:uures by Ixxcnuaung the reversal of niUic oxide aiuvatwm. Following the rcvcrsal of nitric oxide acuvauun at 24' by dithiuthreitol, the particulate entynsc could 64 65
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be re:ntivatcd by a second exposure tu nitric uxidk. Prciniubatwn uf basal p:uticulatc Kuanylatc cycl:ue activity at 37' resulteJ in thc loss uf cnzyn>< respunsivcncss tu aclivauun by nitne oxide or MNNG. This lu» of responsrvcne» that was prevented by the lhiul antwxidarrts was putcntiatcd by the thiad uxidants, dianudc or uxidizcd glu- tathiunc. Alw, the inhibitory effects of the thiol uxidants on cnzymc resprrtsrvcnc» tu activatiun by MNNG were prcvented by dilhiotfuchol. Thcsc results suggest that the activatiun of paniculate guanylatc cyclase by nitric uxidc or MNNG involvcs dte uxiJation of kcy enzyme aul(hydryl groups. BruuKhlrr, J. M. BiacJraericul P/rurrrwcoloYy 31(7):1239-1244, 1982. Frurn tlse f9ugram in Plrarmxulogy, NorUrcastcrn Uhtu Untvcrsitio College uf Mcdr cine, Rootstown. SEPARATION AND DETECTION OF LIPOPROTEINS IN HUMAN SERUM BY USE OF SIZE-EXCLUSION UQUID CIIROMAT(X:RAI'I IY: A PRELIMINARY REPORT This methodological paper presents a rclatively mild anJ quite rapid pnKCdurc lur separating serum lipuprweins fur individual collcction. In a first stcp, human xrum components, irrcluding lipoprotcins, can be rapidly separated by sizc-cxclusitxm "high- perforsnance" liquid chromatography. Then, hpuproteins in fractions of thc cluatc can be quaruitated by eonventional chemical and enzymatic mcthods. Alrernattvcly, rJ lipupnxeias ia the serum arc selectively prcuairred with Jdurmazan dyc, the culunm effluent can be rnonitorod speetmphotomctrically at 580 nm, so that only the Itpupru- Iein components of serum are detected. Samples of purificd low-density IipupuNCmm, so suinaf and analyzeA, provide peak-area values that are proportional to their cunccn- tntion as evaluated by chemical methods. With this tcchnique, the various Iipuprutcut classes can be quickly separated and their concentration estimatcd. Overall, these techniques seem to have potential for developmcnt into analytical and clinical proccdrrces• Brabee, D. L. et a!. Clinica! Chemistry 27( l2):2052-2058, 1981. OlAer suppwf: American Cancer Socicty, Ruturl A. Welch Fowulauun anJ a Nutth Tcxas Statc Uruvcrsity Faculty Rcscarch Grant. From the Depanments of Biological Sctenres and Chcnustry, Niwth Texas Statc Univenrty. Oenton, and the Deparuncnt of Baxlrcmutry, Tcxas Cullcgc uf Chtcu- pathic Metlicine, Ft. Wonh. A NEW SUBCUTANEOUSLY-IMI'LANTABLE Rt:S1:RVUIK FOK SUSTAINED Rf1FJ1SE OF NICOTINE IN T)IE RAT Nicotine has become a widely studied drug for its ph:um.+colul;tial anJ tuatadu- gKal effects. In previous anrmal studres, furced-administrauun metiKilb lur mcixmc included pucntenl injection. adnunrstratiun by mhalauun uf agarcuc suwbf.c, suluMlr rrbun in dnrd.mg watcr, and injection in single Jose% or chronically via Alzct minr pump intu rhe vcntriclcs of the brain, anxmg utJkr.. This paper now rcpurts the successful dcvelupuxnt of a subcutancuusly-implant•able reservurr (ur the sustaineJ rclcase uf nicotine. The tkvice, dubbed INR for Implantable Nicotine Kescrvou, is a sm.ll glass cup sealcd with Silasuc* pulymer. It rckases 3.4 mg of manine per 24 huurs Whcn implanted intu nwtkrately-sizeJ femak Sprague-Dawlcy rals it prrKluccs hh>,><I nicuune Ievels uf 4110-5U0 ng/ml which remain relatively stable uvcr at kasl I8 Jays. INKs ue nuntuxtc, rcpn><luable, incxpcnsive, and aJaptablc (tx pharrrraculogr cal anJ tuRtculu);tcal studtcs iu rats and dher small antmals. 6ryl A.wn, ('. K. rl ul. l'harnwculugy l4rx hemilrry A Brhuvrur 17:183-185, 1982. Frum Drug Dynamtcs Institute, College of Pturmacy, The University of Tesas, Austm. I)li'I7?KMINATION OF TIiL PRIMARY ML'TABUI.ITE OF CIiN'I RAL NI:KVOUS SYSTL•M NUREPINIiPHKINE. 3-MEf1IOXY-4-HYDROXY- 1'lIL•-NI:TIiY1.ENI;GLYCOL, IN MOUSE BRAIN AND BRAIN PEKFUSATE BY HIGH-PERFOKMANCE LIQUID CHROMATOGRAPHY WITH I:LLCI'RUCHLMICAL DETECTION The rate of pn><luctiun of 3-nxthoxy-4-hydruxyptunethyleneglycuI (MHPG), the principal mclabtdnc of nurepincphnne in mam.han brain, has been prupuscJ tu be an irkhcator of the rate of nurepincphrinc turnover. For this reasun, new methiMls of M111'G isulation anJ detecuon arc nnptrn.m in funherrng man's knowledge of the role uf norcpincphnnc in normal and dysfunctional marnalt:ur brain. In the prescM nwlhoJ- ulugy paprr, assays arc described fur the detennination of ptcornolc kveh uf MHPG in nxwsc brain and in the per(usate of an intact mouse txain. High-peAomnancc liquid chromatography with clcctrrschemical rktection yielded a MHPG Jctccuon limit of 0.37 pnwl. This tcrhniquc offers a sensitive and inexpcnsive alternative togas chruma- tugraphy with nuss specuometry, and can be used in conjunction with brain catccMt- lamine determinations. Tuwcll, J. F. and h.'rwrn, V. G. Journal uf C'hruroutugruph)• 223:295-303, 1981. Otheu support: Il. S. 1'uhltc I Ieahh Service. htum llrc S.Iwrrl u( 1'ltarmn:y, Univcnuy u( CuhuaJu. Brwhkr. PRIMARY S-fKl)(TUKIi OF TI11? SIGNAL PEPTIDE OF TR(/1'O1:I.ASTIN b I:Lutm rs a maprr strurtural component of c%mnccave tissues. Irs Wduhk prccur- wu, uultiwlasun, ts cxtract.bk in oaganic xrlvcnts uxl possesses rn cxtcnsrvc:lustcr- tng u( mm/xdar atmnu aad residues in the imnMJute NH:-tcmmnal regnro. Sincc many pndcros secreted from cukaryotre cclls arc initially syntheazcJ wtth aXnal pcpkks, utKr cl.stm wa. cx.rmuwd w see tf il did in fact cImrarn a signal p.ptkk. liadcr scqucrkcs, or signal pcptnks, arc relatrvcly sMut hydruphubtc NIi,-ICrmmal .xten- 66 67
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sions, which arc thought to play a rolc in vcctonal ttanspun uf the naxcnl pulypcptidc. There(ore, thc poasibility that the initial tropuclaaun translation product posscsses a short signal peptide was atamirttd in a cellfrec translation sysacm. In this study, total RNA, isolatcd frvm aortae of I-dayold chicks, was translatcd in an mKNA-dcpcndcnt rcticulocyle lysatc translation assay. The twnslation pruducts wcrc then immumrprca- piutcd and subjoctaf to automatcd radio-scyuencing. Compuison of the NI1; tcrrtninal se.lucnce ofaropoclastin bsynthcsixcd in t)recell-free systcm versus that synthcsizcd in organ culture dcmonttrateG the prescnce of a signal pcptidc 24 amino acid residucs in length. The signal peptrdc sequcnce is as follows: Met-Arg-Gin-Ala-Ala-Ala-Pru- lru-Lcu-Pro-Gly-Val-Leu-Lcu-Lcu-Phe-Scr-llc-Lcu-Pru-Ala-Scr-Gln-Gln. The prc- pundcrancc of hydrophobic amino acid residues as well as the polar residues aJjaccnl to Ilrc initiator nxthionine and the culxwsyl tctmtni (uunJ in llx: signal pclNide is sintJar to that rcfxxtat (or other secretcd puacim. Karr, S. R. and Foster. J. A. The Jourrurf of Hiulugica! Chrmisrry 256(12):5'J46-5949, PJriI. Other supporl: National Inslitutes of Health. From the Department of Biochernistry. University Of Georgta, Athcns. ABSENCE OF SEASONAL VARIATION IN ANTIPYRINE METAIjOLISM Aryl hyr(rocarbon hydruxylase (AHIf), the nwnooxygcnasc systcm(s) that mc- ubolizes bcnzo(a)pyrcne to fluoresccnt phenuls, has been shown to exhibu a strong seasonal variation. The in vivo metabolism of antipyriae, which is also catalytcd by nricrusomal cytochrorne P-450-depcndent munouaygcnases, has been reponcd to he eurrelated with AHH inducibility in human lymphocytes. In the study prcxnted here, an attempt was made to determine whether antipynne metabolism, like AI(H nrctabu- lism, shows seasonalchanges. To do rhis, antipyrine half-life (1)/2) was mcasurcd in 10 nonsmokers and eight smokers in the surmser and the wintcr, the two tirncs of the year Ihu correspcxd to the high and low peaks of inducible AHH actrvity as mcasurcd in lymphocytes. Resulls showed that the mean antipyrine (l/2 dctcrmined in all 18 sub- jects io sunvner was almost identical to that found in winter (x'_SEM = 1U.91) =0.65 and 10.96=0.78 hr)- AHH activity in cultured human lymphocytes (rum the ransmofcing subjects was determinerf in control arxf 3-methylcholanlhrene-induced cells to obtain inducibility ratios of 4.2•_0.56 (SEM) in the sununcr arKl 1.4s0.14 (SEM) in the winter. These results indicate that the scas.Ntal varuurnr in Al ll l itk)ucr- bility in human lymphocytes is not reflected by a correspurxlmg seaxmal vanalwn in anUpynnc met.bolism in vivu. Paigcn, B., Ward, E., Stccnland, K., Bolarw%"ka, W., (irssnrr, T.. Chang, K. 1.., Wood, A. W., and Coruscy, A. H. Clinical Phurnrocology & Thrrupcjaics 3l(2)a44-I50, 1'J2(2. Other srrppor(: National Cancer Institute. From the Dcpartments of Molecular Biology and Expermtentrl Tlt.rapcuucs. Ku.wcll Park Memorial Inslilute, Bu(falo, and the Department of Bwchcmutry atK) Drug Mctabultsm, Hoffmann-LaRoche lnc., Nutley, NJ. ENZYMATIC PICOPERTIES OF IIUMAN GLUCURONYLTRANSFERASE ANl) A SENSITIVE MLTfiOD FOR ITS ASSAY IN A STABLE B LYMPHOCYTE CELL UNE (JDP-glucurunyltransfcrascs arc mcmbrane-bound enzymcs h><atcd chicfly in the cnduplasnric rcticulum of cells and are responsibk (or the conjugation of cndugcnous and xenobi.xic.substanccs containing hydroxyl. amino. thiul, or carbtxyltc functional gruups. Although there is much interest in human glucuronyltrans(crascs, extcnsive studtcs of them have been hampered by dif6cultics associated with procurement of sullictcnt quantities of fresh human cells. This rcport, however, den>.mstrates that a prublic stable ccll htK, such as SNIDU6, capablc of yielding billions of cclls could be used lur such studies. llunKrgcnatcs of SNIt)06 cells have been studtcd. A sensitive rasay pni.'cdutc lur IytuphtKytc glucurunyUranslcra.cc was dcvclu(xd uuhtmg radro- arltvc Icstosleumc as Ihe atcelMUr substartce and 'fLC /ur xpatauun u( the uxuMdqe. 'llrc mctlr.wl is capabk of Jctecting picunwlar quantities of the Irudu.t. The cntymc actrvuy exhtbhcd a broad p11 uptunum, and was subject to acUvauorr by dre dctcrgent l.ubrul W X arwl Mn" ions. Thc axtivity confurrncd to the Michaclu-Mcnlcn ktnctics giving apparcnl K_ valucs Of 0.8 mM and 11 µM, (or UDPGA and testostcrurse, respectively. 4-Mcthylunibcllifcrunc, a-napMhol and P-nnrophcnol bchavcd as cum- pcutive inhibitors of tcstustervnc glucuronidatiun. The results presenled in this papcr indicate that the oscthod couW be used for genetic studies of human lymphocyte glucuronyltrans(cra.se, and that the enzyme is of consequence in dctoaication of exu- genous as well as endogenous substrates. Li, If. C., Portcr, N. anJ Grssnar, T. Enzyme 2g:54-65, 1982. Olher supporr: U. S. Public Health Service and thc New York State Department of f Icalth. From the Department Of fsperimental Therapeutics and Grace Cancer Drug Center, Koswcll Part Mcnrunal Institute, Buffalo. S1111S'1'KA'fE Sl'I:ClUl('lTY OF IIUMAN UDP-GLUCUKONYLTKANSFEKASE IN Cl1L'I*UKED f.YMI'il(X'YTIi.S I luman lym(dtucytcs possess glucurunyltransfcrase artrvuy with a bruaJ range of substruc spcctlmny. For the paper presented here, catalytrr prupertres ul IympMicytc glucuronyltranslcrax were studicd, and activity for the krllowmg subuanccs was documented: teslustcrone, estradwl, phenolphthakin, a-naphthul, 4-mcthylunrhrlh- (cnmc anJp-nrtruphenol. As has been noted before, a-naphthol is regarded as a model aglycur>< sur dk Irrrm Of glucurunyltrausfcrase which.atalyscs glucururriJ.non u( the Ins bulky mxtstcnndal substrates, and Icstusterune as that (ur the stcrurdal aglyiorscs. ll>L results show that human lymphocytes possess glucunnryluanslcrsc aruvHy for both types of aglycunc. Within limitations of work with crude lamugcnares anJ 60 nunutc m:ubatnms, no drl(crence cuuld be seen in apparcnl K. values of l1UP(iA 68 69
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when the vaniws aglycunc substratcs wcrc tostcd. Furtlxn»urc. cumpctit/vc inhlhltion of Icstustcrunc UDP-glucurunyltruufcnsc was ohscrvcd when 4-Incthylumbclli- Icrunc. a-naphthul and p-nitrophenol were used :u the Inhibitrus. Theac data dcrrwn- atrue the similarity of the enzynx(s) accepting the various substratcs. Li, H. C., Porter, N., Holmes. G., and Gassner, T. Xrnabiuricu 11(10):647-654, 1981. Odser srypport: New York State Departmcnt of Health. From the Department of Experimenul Therapeutics and Gracc Cancer Drug Ccntcr, Roawell Park Merrwria) [nstitute, Buffalo. STUDIES ON THE DEPOSITION AND [)ISTRIBUTION Of CATECUOL FROM W[IOLE CIGAR6TTE SMOKE IN BCFI /CUM MICE 7Ac studies rcported herc werc performed to dctcmune the dcpusitiun, dlstribu- tion, and clearance of catechol in cigarette smoke using dclined smuke expusurc curxlitions and a well-characterizcd mouse strain ([)C3FI/Cum). The prcxnce of 1'H/catechol in the smoke was verifred by silica gel chrumatography, high-pcrfurm- ance liquid chromatography, and gas chromatography/mass spectrolnetry. Micc wcrc exposed to 10'b(v/v)2R I cigarette smoke on the Walton Horizontal Smoking Machnrc under standard conditions of 35 ml puff volume, 2 scc/puff, 10 puffs/cigarctte. The deposition and distribution of inhaled catcchol wcrc determined in all intcrnal uswes. urine, and feces. Data showed that ckarance was rxcurring during the lU-min smoke exposure period. Immediately after exposure, over 50% of the radioactivlty was found in the bloal, with 10% found in the lung, and approximately 12% in the respiratory tract. Over 91% of the inhaled radioactivity was found in the urine 120 min aftcr exposurc. Less than 0.5% of the tutal dose was found in the lung at this time. In sumrrrary, these dctaminations show that catechul in smoke is rapidly absrxbcd, rrdistributed and excreted from mice exposed to whole cigarcttc amukc. Hwang, K. K., Sonko, 0., Dansif, D. R., Kouri, R. E., and Hcnry. C. J. (Mrrrobiu- lo6ical Assuciaus) Tasicoluay and ApplicJ Pluunurcolugy 64(3):405-414, 1982. From the Deparrment of Eaperimenul Oncolugy, Microbwloglcal Assoclatcs, Bc- thcsda, MD. EFYl:CTS OF NICOTINE ON UTERINE BLOOD FLOW AND INTKAUTI;KINI: OXYGEN TENSION IN THE RAT Sincc it was known that normal cunccptus dcvclupn>Lnt re4uucs uptlnral Icvcls uf oxygen, it seemed reasorubk to detennine whether mcWlne-induced reducuun In tllerine blood flow concomitantly decrcases the concentrahun of oxygcn within thc uterine lumen. In the study presented here, il was seen that subcutancous injection uf nlcrNlnc (0.5 or 5 mg/kg body wt) resulted in a marked and prolonged reduction in uterine blanl /low and intrauterine oxygen tension in pseudupregnant rals (Day 4). By 1!) minutes aftcr nicotine administration (5 mg/kg) uterine perfusion was reduced by 40'%+, remained suppressed for 90 minutes and returned to the prc-trcatment level by 120 minutcs. Rats receiving the 0.5 mg nicaine/kg also showed a marked reduction in uterine blawd fluw, although the response was slower in onset and longer in duration. Nicotine (5 mg/kg), alsu resulted in a sustaineddccrcase in intrauterine oxygen tension from a control value of 4g.9 S 3.6 tu 22. 2! 2.6 rrunHg at 45-60 minutes and 2 l. 7 s I. 5 mml IK at 60-90 minutes. The frequency and amplitude of Iluctuauon. in intrauterine uxygcn tension were still reduced by 90 minutes after treatment. Thcrchxc, the results ~ of this study indicate that nicotine, in amounts sulhcient to suppress cmbryorllc growth, reduces uterine blood flow and produces a marked and sustained decrease in oxygen tcnan>n within the uterine lumen. The time-courseuf the reduction in intrauter- ulc uxygen avadabdrty pAralkls that of the nicutine-inn[uced decrease in uterine blood Iluw. ll.rnlner, K. E., Goldman, 1[. and Mur-htll. J. A. Journal oJRcprr.Juc-uon and fauliry 63:163-16g, 19g1. Frum the lkparlmcnts of Anatomy ar>,[ Pharmacology, Wayne State University School ul Medlclnc, Dcuuit. SYNTIII:SIS OF Nc)N-K-RF.GION ORTNO-QUINON[•S OF POLYCYCLIC AROMATIC IIYDROCAR[3ONS FROM CYCLIC KETONES 111 thls nrctlwn)uluglcal paper, non-K-rcgiun o-yuinrmes uf pulycyclic arunratlc hydrrKarlxMs arc prepared easily by heating a solution of a tetrahydrrxJn>l with 12 equiv. 2.3-dichluru-5,6-dreyanu-l,4-benzoyuinrrne IDDQ) in dlrraane (rcflux, 24 hrn). DDQ nrx unly oxldlzcs the 1,2-diol muiety a-dlkctrule but ala/ murx)rw-cs the ulclim: dowbk hxmd of t1k r,-quinomc. W hcn this une-stcp crwrvenlun was lirsr dl%Cuv- crcd, the surprisingly facile generation of the o-yuinone was attnbutcd tu the casc of rhnrblc fhNNf furnlatrUrl at the bay-regN)n as deXnbed frX several tctra)lyLhrl .IrerK). I luwcvcr. Ixtxru results uwlicate that the reactiun is not restricted tu thr synthesu uf /lrlllhay-rcglrM U-t1ulMNlcs but b als0 applicable to the preparatnM of bay-re'nNl o- rlulnunes 'I'hu., thrs nrcdl.nl is brNh smlpkr and nlure gcncral than the rc.cnUy puhlUhed %y11tIN %u ol IMIII-K-rc)(H/n o-r)ulMlncs. Ovelall, the synthcuc .rpprrrx•h prc- xntcd tkle ionstrrutcs a new ne/M><[ lur the preparauun of mm-K-regNm rr-yurnuncs that sccros to be generally applicabk. 1'1at1, K. L and Ocsrh. F. Terruhedr„n [.aur•r.r 23(2):163-166, 191/2. Frum tlk Ikpartnlcnr of Tux/culul;y. Institute uf Phannaculogy. Unrvcrsrry of Main[, Marnz, Fcderal Republic ol (icmwny. 70 71
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IMPROVED SYNTHESIS OF ('!)-TRANS-9-lU-DIIIYDROXY-'1•W-DI)IYDKO- BENZOIa/-PYRENE AND OF (=)-TRANS-1-2; DIHYDKOXY•1,2-DIHYDRO- DIBEN-L/a.h JANTHRACENE Non-K-rcgion dihydrodiols of pulycyclic aromatic hydrucarbuns (PAIi) play an important role in the metabolism of PAH. Thcy arc pacursun of dthydnx)wl cpux• ides, some of which arc considcrcd ultimatc carcinogcntc mctabolitcs of PAH. Whtlc the non-K-rcgion dihydrodiols with thc ok6nic bond in thc bay regiun have bcen intensively studial, the biological role of the non•K-regiun dihydrudwls of ben- zo(a)pyrcrsc and dibcaz(a,h1 anthruccnc (6band l3b), whcrc the uk(inic bond is not of the bay tsgion, is )css wel) known. In the present study, improvcd synthetic pathways fur 6b and 13b wcnc devised that resulted in the schemes seen below; a six-stcp synthcsis of 6b (Scheme 1) and cight-stcp synthesis of 13b (Schemc ll). Y~N aN r Y u V~ Qw rc.ult. sub.trnnally. I luwevcr, it way hnally (uunJ that pr)lycychc o•ywnKmc. can hc.unvcmcntly rcduccd to the cuncspunding truns-drhydnxhol% by carrying 4art thc reduction with buruhydridcs in Ju prescncc of oxygr:n. TIk rulc uf uxygcn in the ruluctiun may be rauonaliced by a mechanism presented in this papcr. As a result of thc rcductrun-oxtdauun cycle that takes place when uxygen is avarlablc, pure rruns• drhydruJtol accumulaucs so that it can be isolated in high yteld. lbu ncw method ol reducing K-rcgirm o-quinoncs (and also the corresponding o-diphcnrrls and their acc- tatcs) derived (rum polycyclic arencs represents a signihcant tmpruvement of the synthesis u( K-rcgwn trans-dihydrodiols. Platt, K. L. and Otsc•h, F. SYA?IlESIS: lntrrnutionul Journal ojMarAods in Synthtur Orgunic Chamisrry Nu. 6:459-462, Jwx 1982. Ftunt tlrc Insututc u/ Pharnucology. Section on liiuchemical 1'trarnuculogy, Umvcr• srty uf Marnt, Mainc, FcdcrrJ Republic of Gernuny. ANALYSIS OF NI('OTINE AND COTININE IN 7lSSUE:S BY CAPILLARY GAS C'IIKOMAT(K-iKAP11Y AND GAS Ci1ROMATOGKAPI(Y-MASS SI'IiC'l'KOMETKY In this nrctluafulogical papcr, the developmcnl and application of mct)ods to quantrtatc low levels of mcutinc and cotrninc in tissuc samples arc described. Analyses were pcrfurnscd by capillary column gas chromatography with a spccific nurogcn- phrnplhxus rktcctor and by gas clsrumatugraphy-nuss spccuomcgry. With ck»c struc• tural analogs (or intcrnal standards, high quantitative accurxy and prccrsron were demonstrated for the range of 5-1(l00 ng per g of tissuc. The sensitivity hmn was 2-3 ng/g fur both compounds. The main advantage of these techniqu:s compared to previously published methods is incrcascd selectivity; the other methods were dcvel- opcd (or arralysis of biological fluids and are nut readily ailaptabk to more cumpkx biological matrices such as tissue homogcnates- With the newly developed technrqucs, it was possiblc to pcrfornr a phannacokinetic study of nicotine and cotrnrne in nwusc liver following a single rnuspcriturscal injection of nrcotinc. 'Ibuotpsun, J. A., Ilu. M-S. and Pcrusan, O. R. Journal ojChroawtography 231:53-62, 1982. Frum the School of Phurnacology, University of Colurado, Bouldcr. Since the liuk interest in the dihydrodtols 6b and I3h stcnu partly frum the fxt that their syntheses described in the litcrature ue more difficult than those u( the bcttcr studred iwmcrs, it is hoped that these new improvcd synthcscs may help in funMring study of thc biological roles of 6b and 13b. Ocsub, F. er aJ. The JorrrnaW oJOrganic Chunistry 47:568-571, 1982. From thc Institute of Pharmacology. Section of Biochemical Pharntacolugy, Univcr- sity of Mainz, Mainz. Federal Republic of Gcrmany. K-REGION TRANS-DIHYDRODIOLS OF POLYCYCLIC ARENES; AN [3~~1C(ENT AND CONVE:NIE:NI' PREPARATION FROM O-QUINONES OK O•DIP(IENOLS BY REDUCiION WITIi SODIUM IsOKOHYDRID(? IN Tlili PKESENCE OF OXYGEN K-rcgion trans-dihydnxliols arc inrpun.nr mctabohtcs of (xrlycycbc arumau: hydrocarbons. They arc produced by cpuxidc hydrulasc-nrcdratcd hydrauon of the pnman)y formed arcnc osides. The K-region rra.u-drhydrodrols arc usually synthc- sizad by stcrcosclectivc roduction of the corresponding o-yurnones with cumplcx metal hydrides such as lithium aluminum hydride. When hru attcmpts wcre madc to use sodium or potassium borohydride in asctharwl or ethanol for thc rcducuun of K•rcgrun o-quioones, they w.crc not vcry succcssful. Also, the use of dtffercnt reaction umes and tempaanucs, diffcrcat ratios of the rcactants, and dt(fcrcnt solvents did not rmpruvc. Kh1.A7lONSIIII'S 1)E:1'WiiGN CHEMICAL STRUCTURI: ANl) CIIOLINERGIC ACl'IVffILS O1• FUKMIi'fHIDGS AND POTf:N7]AT1ON OF THLSL A("1'IVI'1'1(:S l1Y ('(IYSOSTIGMINf: The fum><thtdc co,npuunds-(urfuryhrimethylarmxsniurn (fumtcthtdc; FT). 5- ukthyl(umscthuk (5-MNf). 5-hydroxymethyl(unrrthide (S-(IMFT) and 5-:hluru- n>LQtyllunncthrdc (5-CIMbT)-arc potent and stable musearinrc agcnts which arc bcing used in pharmacological investigations where muscarinic sekcuvuy is rcquired. For the studics rcpurtcd here, the ctwrhncrgic activitres of the (uur fumsethidcs wcrc compared on a wpcrfuscd guinea pig rkum pacpararan• Results showed that all four cumpuncnts wcrc kss putent but equally as active as xctylchuhnc (ACh). The EDSOs ( I 72 73
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of thcse coHnpuunds tkcreax in thc following urdcr: 5-CI MNI' > 5-l IM!-T > FT > 5- MFT. The electnm dcrnities around the luran-uxyben atnms ul' these cumpuunds durcaae in the sune order. A careful analysis u( the c(lccts of theae agcnts on the guinea pig ikum rcvcakd that the furnrethides display two acparuc actions: (I) Jircct interaction w the muscarinic receptor to elicit a«sponse, and (2) rclcasc of ACh )rom the prcsynaptic nerve terminal. In the presence of physostigmine, dox response curvcs of these compounds werc shifted to the left. Thcre was approxtmately a two-fulJ shift in the EDSO valucs for the futmc,hide. Like muscarinc, thcsc compounds were not hydrolyzed by cholincsterase (ChE). At kast part of the nwcd lurmethides response was Jue to release of ACh, because thc responses were incrcascd by ChE inhibition. Chaturvedi. A. K., Rowell, P. P., and Sastry, 8. V. R. Pharmacological Research Communicaaions 13(9):829-845, 1981. Othsr sr+pport: National Institute of Child Hcalth and Hunnn Dcvclopnknt. Fruru the Ikparunent of Phannacology, Vandubilt Unrvcrsruy School uf Mcdtcutc, Nashvrllc, TN. CHANGES IN MICROSOMAL MEMBRANE MICROVISCOSITY AND PHOSPfLOUPID METHYLTRANSFERASES DURING RAT UVER REGENERATION Regenerating liver is a good example of a contro)Icd growing ti»ue. During liver growth, newly synthesized molecules of drug-mctabolizing enzymca like eytochromc P450 arc incorporated into the lipid matrix of errdoplasmic «ticulum (ER). There is a morphological association of ribosornal prwcin synthesis with ER rnembrancs. Rcccnt research has shown that decreased membrane micruviscosity facilitates incorporati.ur ofthe enzyme-protein molecules into the phospholipid matrix of ER. The micruvucos- ity of inembranes is altered by: (1) changed ratios of phospholipid to cholcstcrol, (2) unsatwuion of fatty acids in phospholipids, and (3) methylatiun of phospholipids. In a rclated s(udy, microviscosity and S-aderosyl-L-rnethionine (SAM) mediated mcthyla- tioa of phospha(idyklharrolaminc lo phosphatidyl-N-rncthyktharrulaminc (PME) aiMl pLrosptwrdYlcholinc (PC) were mcasurcJ using microsumal mcmbranes of rcgcocrat- iag rat hvas at 6-96 brs after partial bepatectomy. In the nKthylateJ 1>fMrsplwhptJs, the proponioo of PME incrr ased by 3-9% at I µM SAM, anJ, at 200 µM SAM, the proportion of PC decreased by about 5-10% at 12-24 hrs. Two phase Iransitions wcre observed with microsomal membanes between 20 arrd 40'C. In synthetic liposonxa conuining PE. PME and PC, microviscosity decreased when the proportion of 1'MF incrcased or the propurtion of PC decreased. Thcrcfure, aUcrariorrs in ptK»phulipiJ methyltransferaacs and consequent changes in membrane phospholipid mcthylauun may contribute to incrcased membrane fluidity during cell pruhferahorr and tncorltiua- tion of drug-rnctabolizing enzymes into L:R. Jaiswal, R. K., Sarrry, B. V. R. and LArukm, E. J. Phwmac•alogy 24:355-365. 1982. UtAer suppowr: U. S. Public Health Scrvice. From the Departmcnt of Phartrratulogy, Varnkrbtlt University Sch.wrl uf Mcdt.nt<. Nashvilk, TN. IiN1IANCGMENT UF T)11: RESIY)NSIVLiNESS OF THE RAT DIAPHRAGM BY L-ML•T1ilONIN1: AND PLIOSP)IOLIPID METHYLATION AND T11L:IR RELATIONSHIP TO AGING 1bc contractions of hcmidiaphragms of Fischer 344 rats of age 2 to 33 months wcrc measured after clcctrical stimulation of the phrenic rrervc or the muscle in virru. Linccr work has shown tha S-adcnosyl-L-methionine (SAM)-mufiatcd mcthylation of mcrnbranc phospholipids increases membrane fluidity and rcsponsivcness of the nwsclc, and that an increase in the intracellular levels of S-adcnosyl-L-homucystcrne (SAII) inhibits phuspholipid methylation. In the study presented here, the intracellular kvcls of SAM were increased by incubating the hcmidiaplrragm in L-mcthionrnc. L- honx>Lysleinc thiulactone, adeoosine and erythro-9-(2-hydroxy-3-nonyl) adenine, an inhibitor of adenostnc dcaminase. The following results were obtained: 11) Microw- mcs from hcmidiaphragm contained phnspholipiJ mclhyltransfcnscs, (2) 1.- mclhwnutc ukrcaxrd uwx:k tension developed by ekctncal sunwlation u( the muscle or the ncrve. 7Lis uw•rcase in the tension is depcndent uprm the corxcnualion of L- nrcrluoomc. (3) I.Lclcd uxabyl groups were incorporated from labeled L-nxthiurune into )drosphulrpids of tla hcmidiaphragm. (4) Presence of adcnosinc. L-humocystetnc thiolactone and ery(hro-9-(2-hydroxy-3-nunyl)adenine inhibited the cffect of L- nkthiuninc to incrcase tnusck tension and incorporation of methyl groups into phus- phuhpids. (5) Musclc tension developed by electrical stimulalion of the phrcnic nerve or the ntusck decreased with increasing age, and (6) The rnethionine effect was erratic or insignilicant in hcmidiapMagms of old rats (> 15 mumhs). These observaaons indicate that irrcrcasurg the cellular formation of SAM increases phospholiptJ nscrhylr liun and the contractiun heights of the hemidiaphragm upon electrical stimulation. Lkxh of these effects are inhibited by increasing levels of SAH. They also indreate that SAM-medratcd membnnc phospholipid methylation and (ine regulation of inembrane (luidrty in the Jiaphragm may become dcfcrtive with advancing age, and may contnb- ute partly to the functional delicits of the diaphragm. S.urry, 8. V. R.. Owens, L. K. and Janson, V. E. Thc Journal ujPharmac•oluXy and Es prrimcruul Thcnrprwrir•s 221( 3) 629-636, )'I82. Olher support: U.S. 1'ub)ic Ika)Ih Service. National Insutute of Aging, ax) the National Institute of Child Ikalth and )luman Ucvcloprrrcnt. Frum the Lkpartmcnt of Phannaculogy, Vanderbilt University School of Medicine, Na.hville, TN. ' RG(;IILA'1'ION Ol-A('Ifl'1'L('l/Ol.)Ni: RIiLEASE IN 77/G MOUS1i (l:!(I:URLIM 1)Y h11:I111ONINE L:NKLJ')IAL1N AND SU))S"fAN('E 1' Mcduurunc cnkclrlolm (MEK) arM) Subsrance P(SP) may rc);ulate arctykhuhrw (ACh) rclcase ut dre iercbrunr. In the present attempt to study this pniic», nruu.e rcrchra shcc. wcre nWubatcJ in a nMxb)tad Krebs brcarburute buf/cr containing (mrthyl-'l/) chobrK tldundk. lTre slices were filtercJ, washed arx) tuansl.rrrJ to a rwcnbhath sct up fur wperfusww. ACh rclcase was followed by the eltlux u) IabekJ ® 74 75
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ACh and chuline as a function of timc. The rclcasc u(optoid pcptidcs was nrcasurcd by specific radioimmunoauays. The rate of 'H-ACh rclease mcreascd initially for the hrst five min., reached a peak, and then declined expuncntially (half-ume, 35 min.). Electrical fre)d stimulation of thc sliccs during the exponential phax caused a stgni- 6cant increase in ACh release frx 6ve min. Absence of caugcncous Ca" in the perfusion field depressed both spontaneous and cvoked rdease of ACh. When t)u effects of long acting cnkcphalin (D-ala-enkephalinamidc. DALA) and SP on ACh release were studied. DALA (34 nM) decreased the release of ACh (65%) as wcll as Ca" uptake (48%). SP(6 x 10-' M) increased ACh release (35%) and increased Ca' ' uprakc (154%). These observations indicate that enkcphalins may exert a negative feedback control of ACh rekase from cerebral slices by inhibiting Ca" uptakc by the slices. Similarly. SP may exert a positive feedback on the ACh release by enhancing the uptake of Ca •• by cercbra) slices. Sarrry. B. V. R. and Taycb, O. S. In: Dhawan, B. N. (ed.): Carrtnt Status oJCanrru!!y Ar•ung Prprides. New 1'utk: Pergamon Press, 1982, pp. 165-172. (also in: Advancas in the Biusr iani•es: 165-172, 1982.) O4sr support: U.S. Public Health Service. From the Department of Pharmacology, Vandcrbilt University Schcwl of Mu)icinc, Nashvilk, TN. ION BEAM TRITIUM LABELING OF PROTEINS AND PEPTIDES An advanccd techaique for uitiating proteins that yields high specific radioactivt- tics without causing significant changes in biological activities is presented here. In this method, a carefully controlled partick beam composed of T,' and T,' ions and fast T, molecules is accelerated uuo a sample tuget within a vacuum chamber. This beam method has been used to tritiale riboauckase A. porcine pancreatic clastase, themwly- sin, soybean trypsin inhibitor, o,-protcasc inhibitor, and the pcptrde aldehydcs lcupcp- tin and antipain. A(ter removal of all readily cxchangeabk tntrum, the products wcre obtained in 32-83% yields with specific radioactivitics of 18-856 Ci/nwl. The products were carefully charactcrized, shown to be chemically pure, arrd to have complete biological activity. Simple tritium hydrogen exchangc accounts for at least 82% of the reaction pathway with proteins and for 100% of the reaction with the pcptide aldc- hydcs. This method is practical and economical on a reasonable scak, producing high spccifrc tritium incorporation without significant chemical decomposition or structural change to even fragile, high molecular weight materials. Bush, G. A. tt al. (Travu. J. ) The Jorunal oJBiolorical Chewrisrry 256(23):12213-1222 ), 198 1. OrA&i sryywt: National lrrstitutes of Eka)th. From the School of Chemisuy. Georgia Institute of Technology. Atlanu. ISOLATION AND PROPERTIES OF HUMAN NEUTROPHIL MYL'LOPf:ROX1UASE Purification uf human Icukocyte myclupcruxidasc, as descrtbcd in this paper, ts a simple thrcc-srcp procedure involving dialysis of a granule extract agam.t low-salt buffcr, Scphadcx G-75 ctuomatography, and carboxymethylcclluluse chrumatog- ralrlry. The linal product of this purification is homogeneous when examined by acid pulyax:rylanride gel clcctrophoresis and scdinscntation equilibrium ultraccntrifugauun. The molccular weight that was determined here by the IaRCr procedure was 118.000. W ith or without reduction of the protein by 2-mercaptoetharrol, subunits were formed which migrated as a singk band after sodium dodecyl sulfate gel ekwuphrxcsis. The molecular wcight of the apparently identical subunits was 59.000 with rcduction, and 42.()W without reduction. Other general properties of human kukocytc mycloperox- idasc, including amino acid composition, amino terminal sequence analysis, and ab- surjxion spectn, are also reported. Myelopcroxidase, in the presence of hydrogen peruxide and chloride ion, and no otlrcr subsdatc, autoinxtivatcs. Altcr aNnpktiun of the inactivation rcactton, several oxidizable amino acids in the enzymc are modified, arwl the abwiptiun peak at 430 nm disappears. The presence of subsuate of the myelu- pcroardase system (a-l-pnxeina.sc inhibitor), or of high concentration of chloride wn, completely protects the cnzyrnc fronr autumactivatwn. Mathcsun, N. R., Wong. P. S. and Travis. J. Biochemisrry 20(2):325-330. 1981. Other support: National Institutes of Hcalth. Frurn the Dcparuncm of Biochemistry, University of Georgia. Athens. INTEiRACTION OF HUMAN a,-PROT6lNASE INHIBITOR WITH NL?UTROPIIIL MYL:LOPE.ROXIDASE In earlier studies, it has been shown thal a-I-prutcinasc inhibitor (a- I-P1) can be inactivated by myc)uperuxidasc in the presence of hydrogen peroxide and chlorulc ion. Since nryelupcroxidasc may be readily released from neutrophils, its oxidatrvc inxti- vation of a-1-P1 may indirectly result in enhancement of proteolytic destruw:tion of lung trssuc, even in individuals with genetically normal levels uf inhrbition. The aim of this tnvcsttgatiun, thcrc(urc, was to (urthcr investigate the paramctcrs of the myclopcr- uxidc-mcdiatcd uxidativc inactivation of a-1-Pl. These studies, as reported hcre, dcurmsuatc that there is a direct dcpcndcncc on the concentratiun of a-I-PI which becomes saturating at a concentration of about 4.5 µM. There is also a dependence on the concentration of 11,0, to 95 µM, aftcr which increasing cuncuunuuns become incrcasingly inhibitory. Chl.xidc ion is required for myeloperoxidasc uxa)ative action. but the cations Na' , NI1,' , or K' have little effect. Thcrc is a very shup pH optimum at p11 6.2, under physiological NaCI crrncentration, and appruximatcly hal( the ntc of InactlvatNN1 of u-)-1'1 occurs at pf/ 5.9 or 6.5. Sodium dudecyl sulfuc gel ckc- turpMxesis iudtcatcs that a-I-PL oxidized by crther myeloperuxrdasc or N-chlunnuc- cmtnudc ts ruN dtl lcrcnt in si[e from nativc u- I-PI. W hcn oxwfued a-1-PI rs inc ubatcd with prxcme clastase, the a-I-PI is converted to a modified form o1 lower rrwkculu weight. Anutru acrd .cquence analysis cunhmrs these results in that the sequcnce ts dtlfcrcnt Ironr that of native a-I-PI. Ovcrall, from this data and athcr nbxrvarwms reported hcre, it is apparcnt that oxtdative processes directly aflcct the inhibitory actrvuy of a-I-PI nr.abhcation of the rcacuvc-srtc mcUrroninc of this prurern ® • 76 77
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Mathewn. N. R., Wong P. S., Schuylcr, M., and Truris, J. Biur•hcnrisrry 20(2):331-336, 1981. OrBsr srrpport: Nuional lnstitutcs of Hcalth. From the Department of Bioeherrriury. University of Georgia. Athens. INACTIVATION OF HUMAN PLASMA a,-PROTEINASL INIIIBI'fOR BY A METALIAPROTEINASE FROM SERRATIA MARCESCtNS This study was urrdertaken to investigate the interaction of human plasma a,- pnxeirtase inhibitor with a rnctallupr.xeinase isulatcd frutn Sarruria murcrsrrns. Re- sulu presented here show that thc interaction of u,-purtcinax inhibitor with the Senurw pruteinase caused a rapid decrcase in inlribitrxy activity tuwards trypsin which was both corrcenuation- and tirrc-dcpendcnt. To dctermine whether the inactivation had occurred by limited prureolysis, proteinasc-a,-prutcinase rnhibitor incubatrun miatrues were dcrwurod and analyzed by SDS-polyacrylantidc gel ckctrupltoreas. Thcse results show tfuu the prorcinase had rapidly convencd a,-prutunaac tnhtbitur into an inactivc form of lower molecular wcight (48.000 for the malificd funu vs. 52.000 for the native inhibitor). Amino terminal seyucnce analysis indicatcd that the interactwn of the inhibitor and cnzymc was at the reactive site of Ihe inhibitor, with pepr.ide-bund ckavage rcsulting in the inactivation. This prucess may be impun.nt in oecrotic processei uccurnng during bacterial inhltration of host tissues. Virca, G. D., Lycrty, D., Krcgcr, A., and Travis. J. Biochireica ct BiopJrysica Acw 704:267-271, 1982. O(Jur ssyport: National Institutes of Hcalth. From the Deparurxru of Biochemistry, Univenity of Gcorgia, Athens, and the Dcpart- ment of Microbiology and Immunology, Bowman Gray School of Medicine of Wake Farrst Uoiversity, Winaton-Sakm, NC. VI. Immunology and Adaplive Mechanisms CHEMOTACTIC ACTIVITY GENERATED FROM TH[: FII'1'H COM('ONGNT OF COMPLEMENT BY PLASMA KALLIKRf:IN OF THE RABBIT Kallrlrein Iwriticd (rurn rabbit plasma has been shown tn tlk study prc.cntcd h: rc w gcrrcrate chcmuuctic activity for rabbi( neuuupluls (ruru the rabbn's Itlth cumlk)- nent of complernent (CS). The effect on CS appeared to be due to I.allrf.rctn ttsclf atrl nor tu a contaminating cnzyme, because it could be inlubucd by anu-kalhkrcm IgG ur by soybeaa trypsia inhibilor to the same catcnt that ktnin gcncrauun by tIK saunc A.dl1f.rcin ptcparatiun was inhibucd by thcse agcnt.. The chcnwtartic responsc was cunsutcnt with the generation uf a C5a-hkc pcp(idc fronr CS, bccaux thc c(fcct craiW bc partially inhibited by carbuaypcpud'uc N and was related to the gcncratNrn u( a small (-14,(XX) mul wt) fragment of C'S. In contrast, no chcrtkNactK activity could be ~+ 00 -w-i 7-4 J.:nsunsuatul when Inc zynwgcn prcha(Arrem waa tcsta.( wtth CS undcr ufemtcal . crwrditiuns. Chemraactic activity was also generated when rabbit CS was incubated ~ with the 8U,0(J0-M, krcrrr of activatcd Hagcman (aclor, trypsin or EAC423. Nu chcmu- F-~ tactic activity was pruduced when CS was absent from the incubation tntxtures or when intact C5 alune was assayuf. In sum, these results suggest the existence of a novel interaction between the Hageman factor and compkmeM systems which may have ® biulugical rckvance. Wiggins. K. C.. Gtclas, P. C. and Ilcnson, P. M. (Cuchrunr, C. G.) Journal of E.cptrimrnml MeJicint 1311391-1404. 1981. Othersupporr: National Institutes of Ikalth and the O(hce of Naval Research. Urum tlre Ucpattutent ul Inuuwx>[utlM4ogy. Research br.titute of Srnpps C'hnic. La Julla, CA.; anJ the Ihparrnxnt of Pediatrics. National Jewish Hospital. and (kpan- mcnts ul Medicine .nd I'athulugy, Unrvcnuy of Colorado Medical Sclwul, Denver. (;(IINIiA Pl(; HAGtiMAN FACTOR AS A VASCULAR PERMEABILITY I:NI IANCfiM(:NT FACTOR In thts ongoing aucmfH lu asccn:rin the biological ruk of the cuntact (Hagcman Ixtur) systcnt, Hagcman factor was purifrcd frurn guinea pig plasma by successive column chronwtography, and an active Hageman factor. 0-HFa, was prcparcd fur study. The guinea pig Hagcnran factur appeared homogeneuus as a singlc-chrin pru- tcin on pulyacrylamidc gels in the presence of sudium dodecyl sulfate and 1i-mcrcap- tacthanrd. Amtno acid composition of the guinea pig Hageman factor was srmila to that rcptxtcd lur human, bovine, and rabbit Hagcman factors. The punficd guinea pig Hagcnwr factor, as wcll as guinea pig pla.sma, showed strong clotting ome correcuurr activity in liagcman-lactur-dc/icicnt human plasma. The activity could be bhrckut by the IgG fraction of antiscrums againsl guinea pig Hagernan factor raised in rabbits or a guat. The concentration of Hagenun factor in guinea pig plasma was dctcrnuncd to be 120 µg/ml by quantitative radial imnwnodiffusiun assay. When ~-IIFa, the 28,0(1(J- dalurn active (urm of Hagcman factor, was prepared from guinea pig Hagcman factor by treatment with pla.nu kallrfucin, (!-F1Fa caused an increase in vascular penneahd- uy when i*ctcd into guinea pig skin at concentrations as bw as 3. 10" M. This tncrcaxd permeability was shurt-lasting, and the permeabiluy enhancement xtrvuy uf (3-11Fa was inhibited by pretreatment of {i-HFa with dnwpruphyllluurup(wsphatc. According to the autMrn, rt may be concludcd, therefore, that acbve Hagcnun factor in the tntcntiual spacc uf guinea pigs acts as a vascular permeability frtor of lu greater potency than bradykintn. YamanuKU,'1'. arnl C'9ahrunr. C. G. Arnrrviun Juurn~) uf Y~nliulagy 105(2):1(r3-173, 1981. (lrhrr supporr: NauANsal ansututcs uf Ilcaltlr atwl the O(h.c of Naval Kcxuch. I-rum thc Ikputnknt us linnwn.ppathulugy, Kcscarch Instttutc uf Scnpfn ('Ima, 14 Julla, CA 78 79
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MODULATION OF POKEWEED-MITOGEN-INDUCI:D IMMUNOGLOBULIN SECRETION BY HUMAN BRONCHOALVEOLAR CELLS The cffects of pulnroauy alveolar macropbagcs (PAM) on immunuglobuhn (lg) secretion were investigatod using autologous periphcral blood lymphocytcs as the indicator population and pokeweed mitogen (PWM) as a morwcyte-dcpcndcnt puly- clonal B-ccU activator. Broocboalvcolarcc)Is (BAC) from seven nonsmoking subjects suppressed the response to PWM by unfractionatcd autologous pcriphcral blood marsorsuclearcclls (MNL), whercas low concentrations of BAC partially reconstituted thc rcsponsc of morocytadcpleted MNL to PWM. Thus, it could be sccn that BAC could modu)ate PWM-irrducod Ig sccretion in different ways, dcpcnding on the pres- crre or absence of rsxrrsocyla in the rrononuckar cell population. The suppressor activities of BAC were not abrogaltd by prior irradiation and were only partially rsversed by thc addition of indornethacin to the cultures. Howcvcr. poor disruption of BAC completely abolished their suppressive functions. Suppression of PWM-induccd Ig socrctioa is prubably rrsodiated by iatact, radiuresisunt PAM. Lawrence, E. C.. Theodore, B. J. and Marsin, R. R. Anrerican Review of Respiratory Disease 126:248-252. 19112. Or(ser syPport: Amcrican Lung Association and the National Instrtutcs of Hcalth. From the Rockwcll-Keough Pulmonary Immunology Laboratory and the General Clinical Research Centcr of the Methodist Hospital, and the Depannacnt of Mcdicinc, Baylor College of Mcdicinc, Houston. DEFECTIVE IMMUNOGLOI3ULIN SECRL•T1ON IN RESPONSE TO POKEWEED MITOGEN IN SARCOIDOSIS Several previous studies of sucoidosis have indicated a dichtomy between cn- hanced humoral immune functions clinically and dcfcctivc in vitro B cell respunsive- ncss, which wggcstod some dison(cr of immunorcguluwn. In the study prcscntcd hcrz, it was found that polyckxoal immunoglobulin (1g) sccretory rcspunse to pokc- woed mitogen (PWM)-a plant lectin which rcquires the prescncc of both nwnucytcs and T ccUs in order to triggcr B cclls-was dc(cctive as well. Specifically, in vitro irnmursoregulatioo of Ig secrction was studied in 21 patients with sarcoidosis. While pcriphcral blood morrorurckarceUs from normal individuals respundcd to PWM with a 10-fold or greater irscraaent in lg-secrcting cells, cells frorn sarcoid patients failcd to rupond to PWM at any catccntration employed. More monocytcs wcre found in sarcoid rnonornrc)ear cell prcparations (44.8s 2.U`bvs 30.4 ± 1.4% in normal donors). but rtnsova of raorsocytcs improvcd the responsc to PWM in only (our patients. Moaonuckarcclls (ran seven of 19 paticnts suppressed Ig secretion in co-cultures with normal donor cclls. Patients cxhibiting cacessivc supprcasur cell funcuon were uldcr, with Iwtgu standing and ku clinically active disease than axr-wppressing paucnts. Mueocytc removal reversed thc suppression in only (rwr uf the suppressrx paucnts, but excessive wppressor rrsonocytc function was laltr denrunsuatcd in twu sar.urd paucnts whose cells initially did not suppress Ig secretion when cultured with nururd cclls. Wbik the immunological defects in sarcoidosis may be cornpkx, hctcruguaous And dynamic, thcsc Aata suggest that suppressor moncxytcs, when presen: rn sarcuidusis, may have rkvcluped xcorxlarrly. M DD .-i L.awrrnre, E. C. et al. ri Cliniral and E.cperrmental Immunology 49:96-104. 1982. Other support: Anr.:rican Lung Association And the National Institutes ol Health. From the Rr><kwcll-Kcough Pulrnonary Immunology Laboratory and the General Clinical Research Center of Thc Mcthodist Hospiul, and the Department of Mcdretnc, Baylor College of Mcdreinc, Houston. NEUTRAL GLYCOSPIIINGO(aPIDS OF I(UMAN ACUTE LEUKEMIAS A nkthrHkrlugy was intruduccd in this study which combines the scnaiuvuy of high pcrfunnancc liquid cluonsatography wrth the specificity u( cxo- and crKluglycoat- daacs to study the neutral glycosphingolipids present in the malignant celb of 10 paucnts with acutc Icukemia. Results showed that acute leukemia cells contain very little ur none of the mure complex neutral glycosphingolipids that arc found in nonnal Icukucytcs or chronic kukcnaia cells. Lymphoblasts, in panicular, are rich in neutral glycosplamguhpids with only one or two carbohydrate units. The rnusl significant /inding of thrs study was that, in contrast to normal leukocytes and chronic kukcmia cells which have a single predominant teuausykcramide species, acute leukemia cells (9 out of 10 patients analyzcd) were found to have significant amounts of both globo ((ialNAc(il Y 3Gahrl Y 4GalPl Y 4GIc(il Y kcramidc) and ncolactotctrausy- kcramrdc (Gal(11 Y 4GIcNAcj3l Y 3GalPl Y 4Glcal Y kcramide). Thcx results uadtcatc that the cumpusitiurr of neutral glycosplsingolipids in acute leukemia cells differs signilicantly frunr that found in normal or chronic kukemia cells. Lcc, W. M. F.. Wcstrick, M. A. and Macher, B. A. The Journal ujDiolugrral Chemistry 257(17):lOD90-1l)095. 1982. Other support: National Institutes of ((ealth, National Cancer lnsutute, l.cukcmta Research Foundation, and Cancer Research Funds of the University of California. From the Cancer Research Institutc, University of California. San Francisco. GLYCOSPHINGOLIPIDS OF NORMAL AND LEUKEMIC HUMAN L(iUK()CYTES Studies on neutral glycosphingolipids and gangliosidcs of normal And kukcrntc human kuktxytcs were reviewed (or this ptrsentatiun. It can be seen here that two metlKxk)lugical appnranccs have been used tu determine the structurc And dratnbutum of glycusphingolipids aniong human kukrkytcs: (1) those that utrlruc chemical and cnzynuUc touls lu r)ctcnnine the compkte structurc, and (2) tMr.e that rcly rwt rrwluect as.+ys uf dctcctx>,t 'I Ix hKmcr nK ItwKla alhrw unc lu.»ry,n a umyuc auurtwc tu c.ih cunyxrund, but they havc twa rLsadvantagcs: (1) they rcyurrc rclauvcly large yuanuucs uf uratcrrala anrl (2) nniwu cuml><Nrcnts nray be lust during the paK'c» of prcparrng Iwmwrgcnuus .urulnrunk)s fur analyats. lrwLrect methods which have h..n used arc scnsdive arrd raprd, .uk) allow onc to cumparc casily several samples, but Ury y have the ~ /-~ U W 80 81
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Jis.Jrurtagcs of being indirect, with structures asstgncJ on the basts of compariwn with uartJards. Examincd for thrs rcvicw were: (a) the glycr»phinguhpiJ cumlxrsiuun of varwus Icukocytc populatiuns. (b) the diffcrcnccs in glycusphinguhpiJs found anwng subscts of these cclls, (c) the possible use of thcsc compounds as markcra of Jiffercntiatiom, and (d) the changcs in glycosphingufiptJ composition that occur with kukcmogcncsu. Aluchrr, B. A.. Lcc, W. M. F. and Wcstrick, M. A. Molecular and Callular Biochemistry 47:81-95, 1982. OtJur sypport: National Insututu of Health, National Canccr Instdutc. Leukcmia Rcscarch Foundation, ruwd Cancer Researtih Funds of thc Univcnrty of Cahfurnia. From the Canccr Rcsearch Institute and Depanment of Pharmaceutical Chcmistry, Univcrsity of California, San Francisco. NEURON-SPECIFIC ENOLASE AS AN IMMUNOCYTOCHEMICAL MARKER FOR TFfE DIFFUSE NEUROENDOCRINE SYSTEM IN HUMAN FL'TAL LUNG Ncuron-spccific coolasc (NSE) is an isoenzyme uf the glycolytic cnzyme crrulax. which was originally considered to be rcstricted to ncurons but has recently bccn shuwn to occur in sorne APUD cells. In this papcr, the localization of NSE in thc Jif(use ncurocrsdocrine systear of human (ct.l lung is reported. Specifically, NSE-positive cells, singly or in groups, werc demonstratcd by antiscra raised to human or rat NSE. lmmunosuincd serial sections indicated that NSE-positivc cclls could also contain bombesin and /or SHT-like inrmunorcactivity. At kast three Jifferent ccll types werc identifrcd conuining (1) NSE, SHT, and bumbesin, (2) NSE anJ SIIT, and (3) NSE a)one. After close considcratioa of thc matcrial prescntcd hcrc, it appears that NSE is a uscful rnartrer of the ncuroersdocrioe systcm in thc lung as well as in onccr ussues. Alw, the lack of altcrrutive simpk and reliable tcchnpues capabk of idcnti(ying both cells and naves mcans t)ut the irnmuaocytochcmical loca)izatron of NSE is a valuablc tool for the strrdy of dcvcloprrrcnt, physrology. and pathology of this system. Wharton, J., Polak, J. Al., Cole, G. A., Marangos, P. J., and Pcarx, A. G. E. The Jarurra) of Histoduwri.ury and Cytochenristry 29(12):1359-1364, 1981. From the Department of Histocherrustry, Royal PostgraJuate Medical School. Lon- don. England, ard the Clinical Psychobiology Branch. National Instuutes of Health, Bcthcsda, MD. IgE-DEPENDENT RELEASE OF LEUKOTRIENE C. FROM ALVEOLAR MACROPHAGES Slow reacting substances (SRS) have becn shown rcccntly to be a 1'anuly of pcptidolipids called kukotricncs (LTC.. LTD, and LTE,) thar arc dcnveJ Irum aracht- donic acid and ue potcnt brunchoconstsictun iw vivo and rn vitro. In tlw s1uJy ptc- sentul herc, rat alveolar rnacruphages age shown tu trc thc cells tcslwtustbk hK rckasiag dre JRS. Tlse ability u( Utesc macru/.luges to telc..c SRS ws u•.rcJ tniualy by incubatingccll suspcnsiuru (ur lU uun. wrt/r I p.utul ul calcrurn wtwtpMwc A231ri7, in the prescncc uf 5 x IO 'M L-cystcinc. In subsequent cxperin>Lnu. ccll suspcnsiuns wcre sbmulatcJ with pun6cd rrKwse munoclunal anti-DNP (dinitn>plknyl) IgE anti- body and DNP-Iruman scnun albumin. Rcsults of thesc capenmcnts slww that (1) rat alvcular macnoplugcs release SRS when stimulatcJ non-specihcally by the calcium wnuptxxe A2311S7 in the prcscncc of L-cystcinc, and (2) IgE antibody and appruprutc antigcn cause alvcular macrophagcs to rekase SRS leukotrxnc. LTC.. This dcmon- stration that rat alvcular macrophagcs rckase SRS by an IgE-dcpcndcnt mechanism raises the possibility thallgE-dcpendena rckasc of mediators by alvcolarmacrophagcs nuy have a role in asthma or other immunologically mufiated lung diseases. Rankin. J. A. ct a!. (Rcynolds. H. Y.) Nature 297:329-331,1982. Other support: National Inuiuucs of lkalth and Huffman La-Rochc Inc. From the Department of Medicine and Pharmacology. Yak Univenity School of Mcdicrnc, New Haven, and the John B. Pierce Foundation. New Havcn, CT. MONOCLONAL ANTIBODY ANALYSIS OF COMPLEX BIOLOGICAL SYSTEMS: COMBINATION OF CELL HYBRIDIZATION AND IMMUNOADSORBENTS IN A NOVEL CASCADE PROCEDURE AND ITS APPUCATION TO THE MACROPHAGE CELL SURFACE In this wphisucated methodological papcr, a procedure is dcxnbeJ which greatly simpldics the collection of monoclonal antibody (MAb) libraries JtrccteJ towarJ individual compuncnts of complex biological systenu. F.x the study rcporreJ hcrc, removal of previuusly recognized antigens with immunoaJxxbcnt columns was cunt- bincd with cell hybridization in a cascade which restricts the immunizing stimulus tu previously unrecognized antigens. Specifically, in Ihis report a cascade pruccdure was explored in connection with the identification of further macrophagc-specifrc antigens. Pcntoncal cxrKfate cell mcmOrurcs were detergent solubilized, and the previously iJcntificd corrurwn kukocyte aruigen and hcat-stabk antigen which are shared with pentonul exudate cells and lymphocytes were removed with MAWinununoadsor- bents before imrnunization for the hybridization experiment. Removal of the antrgens was cunfinned by radioimmunoassay and by the scrological response to immunizarion. Scrum antibodies to specific antigens were also measured to compare the efficacy of this proceslurc to immunization with either whole cells or MAb-coatcd cells. Two previously unknown macrophagc-specific antigens of 32.000 and 110,Q00 M, were iJcntitiod here. According to the authors, the prccedurc can be exterxkJ by arrangtng further immunoadsurbent depktions and cell fusions in a cascade series and is rcadrly applicable to the nwnoclunal antibody analyses of many other multrcompxrcnt brulog- ical complexes. Springer. T. A. IhrJ..urn..l./N,u6.grral C'hnnurry 23(0):31413-3g39, 1981. (hhrr sapporr: (/, S 1'ubbc llcaltb Savr:c. 1'rutu Ihc tk/wlMcnl ul I'atl10bgy, HarvarJ McJW,6l School. Buuun ca oO ~ ~ • ~ h U M 82 1/3
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MAC-I, 2. 3 AND 4: MURINL MACROPIIAGE DIFFFI:RI:NTIATION ANTIGENS lDE:N-I'IFIED BY MONOCLONAL ANTIBODIES The Kohlcr-Mibtcin mycloma hybrid tcchniyuc, which can cnable the isolation of a morroclonaJ antibody recognizing a single antigcnic rktcrnunant frum an intual highly complex antigen, has given great impetus to the analysis of cell surface com- pkxity. In the paper prcsentaf hcrc, work dorre in the author's laboratury using this lechoiqrrc for the identi6catirw and study of macrophage antigcns is rt;vtcwed. Four antigens-Mac-1,2.3 and 4-have been identified by the corresponding n>.nxxlonal antibodies. MI/70. M3/31. M3/38, M3/84 and M3/37. These antigens all appcar to be oa the macrophage cell surface on the basis of fluorescent and "'I-labcling. "S- methioriae incorporation into the polypcptidcs by the adhercnt fraction of thiuglycol- late-induced pcritoneal exudatc cells also suggcsts these antigens arc synthesized by macrophages. The four different antigens defrned in these studics arc prescnt on rnacrophagcs. but not lymphocytcs, demonstrating the distrnctivcness of macrophage cell surface architccture. Currently, the expression of these antigens on macruphagcs induced by other means and in different anatomical locations is being invesugated. The rrxxrockxral antibodies are also being used as probes to inhibit a pancl of macrophage functions. In this way, it should be possible to link the molecular structures described hcrc with specific macropfssge ccll surfacc activities. Springer. T. A. In: Fwster, O. (od.):Ileturojeneiryojmononwcleurplwgrx•ytas. New Yurk: Acnlenric Press. 1980, pp. 37-46. Orlsrsrr,Pporr: U. S. Public Heafth Service. From the Department of Pathology. Harvard Medical Schuul. Boston. A SHARED ALLOANTIGENIC DETERMINANT ON Ia ANTIGENS ENCODED BY THE I-A AND 1-E SUBREGIONS: EVIDENCE FOR I REGION GENE DUPLICATION It has been known for a while that the I region of the major histocumpatibilhy complex contains genes that eontrul immune rrsponse and imnwne suppression to certain antigens and differcnt dcterminants, and studies on these genes have led to the definition of a number of 1 subregions. In the study presented here, two nt monuclonal antibodies (MAb). MS/114 and M7/81, which have a very unusual type of crossrcac- tive specificity for murine I region proJucts, arc charactcrized. These MAD detect polymorphic dcterminants present on B cells and actiyated T lymphocytes from nuce carrying the H-2•, H-2', H-2', li-2•, and H-2•haplotypes but not from mice carrying the H-2' or H-2' haplotypes. Aruigenie site number determinations showed that the pusitivc hapbrypes can be divided into two groups. Mice bearing the H-P, H-2'. and H-2• haplotypes express a high number (40,000 to 80.00U) of antigcnic sitcs per B lymphu- cyte, and MAb plus complement can lysc B cells frum these mice. In ctmtrast, micc bearing the H-2' and H-2' haplotypes express a low number of antigcnre sitcs. Splccn cells from mice carrying the latter haplotypes are not Iyscd with MAb And complemcnt. Genetic mapping demonstratc.d that high and low expression map to the I-A And 1bE subrrtgions, respectively. The MAbdetect an la specificity on 1-A'. I-A',1-E'. and 1•E' molecules. These observations were conlirnxd using sevcral diflcrcnt cx(annrcnlal approaches, i.e., cytotoxicity, fluorescent staining, curnpcuuve mlubiuuo of MAb bnulutg, arw/2-dinr.nsiunal gcl cscctruphoresis of immunoprccipiutcs. KcsulU ul this study provide rmmunrdugic evidkncc for homology between I-A and 1-E antrgcns, And herrcc lur gcrrc duplication within the I region. Ilhattacharya. A.. IAKf, M. E. and Springn, T. A. The Journal ujlmmunulagy 127(6):2488-2495, 1981. Orlur suppurt: U. S. Public Hcalth Service. Frum the l.bixatury of Mcmbranc Immunochemistry, Sidney Farbcr Cancer Institute, And the Dep:utmcm of Pathulogy, Harvard Medical School, Boston. NATUKAI. K11.L.IiH ACI'IVITY IN THIi PERITONEAL EXUDATES OF MICE INF1iCfED WITII I.ISTERIA MONOCYTOGtNES: CHARACTERIZATION OF TIIE NATURAL K11.1-EK CELLS BY USING A MONOCLONAL RAT ANTI-MUKINE MACROPIIAGE ANTIBODY (M1/7U) Natural kilkr (NK) cclls arc nwnunuclcar cclls of disputed lineage that krll crnarn destructive cells in the body. In the papcr presented hcre it can be seen that mlccerun with l.isrrnu munoH yruArnrs (LM) Ieads to the gcneratwn of NK activity in pcntuncal cxudatcs. Spcci/ic.lly, rnaxinrum expression of NK activity first uccurrcd on day two and remained high until day six after initial exposure to LM. When nylon wool nunadtK:rcnt peritoncal exudate cells were examined by a singk-ccll cytotuxichy as- say, the number of cells binding to YAC- I target cells increased after infection as did their individual lytic capacity. A nrxroclunal rat anti-murinc macrophagc anubkxly (MI/7U), previously shown tu recognize human NK celb, can be used al>,r as a mar kcr for murine NK cells. Utilizing MI/70 and the /luorcscence-activated cal wner, scicc- uun of MI/7U-labcicd mononuclear cells kd to the enrichment of both NK and anti- brxly-dcpcnrknt ccllular cytutoxicity. These MI/70-pusitive cells had a distinctive nwxplKrlogy and contained granules on Wright-Gremsa staining. They were not phagu- cytic, did not conlain nonspecific esterase, and lacked surface 1-A'. IgM dctcmunants, complement rcccptura, and high levels of Thy 1.2. Holmberg, L. A., Springer, T. A. and Ault, K. A. Tht Journal uj/mrnunulugy 127(S):1792-1799, 1981. Other support: Nauunal Institutes of Ikalth. From the Dcpartn>tnt of Pathology, Ilarvard Medical School, Boston. MA('ROPIIAGE I)IFFERtiNTIATION ANTIGENS: MARKERS OF MACHOPHAGE SUBPOPULATIONS AND TISSUE LOCALIZATION 71K ch:rractcristres ol' hwr dutmct antigens which arc present rnr m.rruphagcs, hut not lymfrlMxytcs, :rrc rcvtcwcd in this paper. In adddwn, two appbc.rtnrns ol antr- mxrrrphal;c mruwwhnral rcagents-Ihcu use in phcnotyprng macrophagc subpupu- latrons And in idcorrlrcauun of nracarphages in ti» ue xctruns-arc Jcxnb.d here. '11be waprr sectiuns of thrs paper are devoted to MAC-I ANTIGEN. IMMUNO- AI)S()KIIIiN'I'-CI:1.1. IIYIIKIUI"LATIUN CASCADES, MAC-2. 3 AND 4 ANTI- 84 . 85
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GGNS. And IDENTIFICATION OF MACRUPIIAGIiS IN'CISSUt: SL'.C1'IONS LlY INDIRECT IMMUNOFLUORESCENCE. Overall, four tnrcruphagc anul;cns whh distinct Mr and tissue distribution ara idcnNtied in tho work. Two ul-thcx, Mac-I And Mac-3. arc synthesized by all macruphage wbpopulauuns cxamincJ thus lar. Huw- ever. Mac-2 seems to be prcfercntially asxocutcd with thiuglycullatc-didtcJ pcrilu- ncal macnophagcs. Ia antigens show a dtffcrcnl paacrn uf cxpresstun. Thercfurc, nucrophagcs can be defincd into subscts with datmct antrgcnic p)xnutypes, as is the case for lymphocytes. Jpringrr, T. A. and Ilu, M-K. In: Mitchell, M. S. and Oettgcn, H. F. (cds.): IlybrnJumus in Cuncrr Dmgnusis unJ Trcumrenr, Ncw Yorit: Ravcn Press, 1981, pp. 35-46. Ol4sr srtipport: U. S. Public Hcalth Service. From the Dcparuncat of Pathology, Harvard Mcdical School, Boston. RAT ANTI-MOUSE MACROPHAGE MONOCLONAL. AN-rIBODIES AN[) THEIR USE IN IMMUNOFLUORESCENT STUDIES OF MACROPHAGES IN TISSUE SECTIONS The characteristics of five monoclonal aruibalies (MAb) to macrophagc uuigens arc sumnwizod ia this paper, which also contains a description of the use of uoe of dresc MAb (or the lacalizatioa o( macrophagcs in fruzen splecn sections. Of the live rat rnwocloaal aatibodies to mouse macropluge surfacc antigcns that wcrc tkvclopcJ in the authors' laboratory by the myebma-spkcn cell hybrid tcchnique of Kohlcr and Milsleia. MI/70, which recogaizes a phagocyte-specific antigen. Mac-1, is the nwst cstcasivcly studiod. Twoorber antibodies, M3/31 and M3/38, precipitate a pulypep- tide turnod Mac-2, which is also characterized here. In the related study. anti-Mac-I was used to staia rnacrop(sages in spken sections because Mac- I seems to be expresacd on nucrophagu irrespective of their state of differcntiation and activation. To alluw aligomca of arcas with Mac-I' cells with T-ckpcrsdent arcas of the spleen, adjacent sections were suinod with M5/49, an anti-Thy-1 MAb. Results showed that T IympMr- cytes in the periarteriolar lymphatic sheath arc intensely stained by ami-Thy-1 MAb. In coarrast, (ew, if any, Mac-1 ' ceUs can be seen in thesc T-dcpcndent areu. The simple mcthod for thc {ocalizaion of macrophagcs described in this publication can be easily extended to orber studies, especially with anti-Mac I, which stains all macruphagc subpopulatioas ezamiaed so far. In vicw of the vast body of information that can be gained from anatomical localization of lymphocyte subpopulations, these studies should provide much insight iruo the function, differcntration, And uruugcny of asacropfuges. )b. M-K. and Springcr, T. A. la: Haauncrling, U., Hammctling, G'and Keamcy, 1. (cds.): Munurlorwl anubuhrs and T cell bybridomas, New Yortc: Elsevicr/North HollanJ Biuukdreal I'cc» , 1981, pp. 53-61. OrAsrsrypport: U. S. Public Health Service. From the Laboratory of Membrane Immunochcmistry, Sidney Farbcr Cancer lustuutc. llarvarJ Mu(ical Schuul, Boston. ~ MAC-2, A NUVLa. 12.(1(lU M, MOUSE MA(-KOI'HAGSi 00 SUIINOI'L11.A'1'IUN-SPGCIFIC AN-1lGEN DEFINIiD BY MONOCLONAL V.( ANTIBUI)IES 4-4 The blUlhl'ImCaI lh:IractcrlLatlUn and cell distribution nI a 32,(Xtt) M. anttycn, tcrnxd Mac-2, :uc presented in this pal><r. Mac-2 is synthcsucJ by And cxpresscJ on a the surlacc of thiuglycullatc-chchcd nucruphages as shown by I"SI-n>Llhiomne and ~ "'1 labcamg. Unclicitcd peritoncal macrophagcs and macrophagcs elicited by procc:rse C /xlxunc, Con A, U'S. and Lrsrrrra munuryrugrnes are either only weakly positive or ncgativc. llkre(urc, Mac-2 cxpressiun is induced only by strong inflammatory stimuli arxl appears spccilic for munonuckar phagocytc subpopulations in a distinct stage of = differentiation. Results of saturation binding experiments show that thioglycollate- ehciteJ macruphagcs express 1.7 x 10' Mac-2 sites/ccll. Thioglycollate-elKited marns- phagcs are strongly abwrptive for °'I-labcled M3/38 MAb. Cell suspensions from spkcn, b.me marruw, thymus, and peripheral lymph nodc arc > 9911, Mac-2 ncgative by immwwfluuresccnt flow cylumetry. In contrast, thioglycullare-elrciteJ marro- phay,cs arc > 96% strongly positive fur Mac-2. SDS-PAGE of J"SJ-mcthirusinc- labclal Mac-2 shows that thioglycollatc-clicited macroplragcs synthesize 10- to 30-fuld more Mac-2 than othcr pcritoneal macrophagc subpopulations, whcrcas all types of pcritoncal macrophagcs synthesize and exprcss on their surfaccs sinular amounts of the Mac-1 antigen. Mac-2 antigen is, thcrt(ore, induced in macr.rphagcs only in response trr specific diffcrcntiative signals. I lu, M-K. and Springtr, T. A. Thr Juwnul ujlmmunolagy 128(3):1221-1228, 1982. Orbar support: U. S. Public Health Service. Frorn the Laburatory of Membrane Immunochcmistry, Sidney Farbcr Canccr Inststutc, Boston. ONTOGENY OF MURINE MACROPHAGES: FUNCTIONS RELATED TO ANTIGEN PRESENTATION Imnwturc macruphagc function contributes to the increased susccptrbtfity of neu- natcs to infctuun. In this paper, the immaturity of nconatal macrophage function is dissected into antigen prescntation and three different cffecrorcumpuncnts: cytotoxic- rty, anttgcn uptake aa/ catalsolism, and the production of the IympMrsumulalury nxrlcrulc intcrkulin-1 (alw called thymucytc mitugcnic protein or lymphr><yte-ac- tiva(mg factor). The uptake anJ catabolism of'°L-IabckJ Lrsrrriu nwnoK.rrugenrs were equivalent in nracruphagcs from adult and neonatal nricc. Huwcver, rnteracuoms Lx- Iwccn rnarruph.rges frum neonates, hcat-kilkd Lisrrrru urganisms, and tmniunc T lywplnnytcs were impaircJ, and no cytucidal nucrophages capable of killing rumor cclls were generated. Previous studics with ocils ft+an adult micc had established that the Jevchsprtx:nt of cytu.kla macrophages required la-Acaring, antrgcn-prcuntrng macrophages and histocumpatibthty at I-A between macruplugcs and T cclls. To crrcunwcnt this requirement for anngen-preaenung nucruphagcs- an as.ay was used in which IyaoplHdunc was :rkkd directly to the macruphagcs frorn nrunucs. Strrwtg cytrwrJal activtty resulted. Thus. these stuJres showed thal nurruphal;cs frum ncu- natcs Ixcscnt uwben fMxuly but can acyuue cytucidal luniuom IxuvrJeJ that thc need 86 87
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frx antigcn-prescnling function is bypassed. Suuilrr cutKluswns were rc.rchrd lur the sccrctkrrr of intcrkukind. In esscn.e, all ttk data prcuntcd Ikrc urdiiatc th.n tIk inrpainrrcnl of a numbcr of rnacruphagc functions in the rrcunatcs is due to a rcduccd numbcr of la-pusitivc rrracrophagcs. Lu. C. Y. and Ununne, E. R. In/ecrion and Irnrnaniry 36(I):169-175, 1982. Othsisrrpporf: National Inuitutes of Health and the March of Din>ts. From the Deparuncnt of PaUwlogy, Harvard Medical Schous, Boston. CONTROL OF MACROPHAGE Ia EXPRESSION IN NEONATAL MICE- ROLE OF A SPLENIC SUPPRESSOR CELL As rcpurtcd in this papcr, the control of macrophabc expression of 1 rcgiun- associated antigens (la) in neonatal micc was studied by comparing resprmscs of neonatal and adult micc to immune vs. nonimmunc stimuli. Adults gencratcd pcrnu- nuJ exudatcs rich in la-bearing macrophages in response to i.p. injcction of live Llslrria rrwnocywgenes, Listcria-immunc T cclls, and hcat-ktlled Lisreria, or a soluble mcdixor termed rnacrophagc la-rccruiting factor (MIRF). Neonatcs failed to respond to thcse stimuli. In contrast, both neunates and aJults gencratcd la-ncgativc pcritorseal exudates when stimulated with thioglycollatc. There arc thrcc major new points that came out of thesc studies: (I) neonatal mice not unly have a dcfcct in their basal number of la-positive phagocytes but also fail to respond to tlu: imnwnc stirrruh Uurt generate exudates ennched fur these cells; (2) thcre is a suppresxx system ulxxtat- ing in the neonate capabk of signifu:anUy dampewng the rccruumcnt of la-pusruve macrophagcs-this suppressor system is also opcrant in sun>c :uluh tissues such as bone marrow and the peritoneal cavity; and (3) the suppressxx mcchanism invulves, at kast, the phagocyte system by way of an indumcthacin- and aspinn-scnsrtivc step. Ovcrall, it appean that this pAagocytic line autorcgulatcs its surfacc expressron of la tn both neoruul and adult mice. Since this mechanism becomes panicularly pointed during cul y development, it could contribute to Qre lack of inrn~unity durtng ontugeny. Snyder, D. S., Lu, C. Y. and Unanwc. E. R. The Jownal aJ/nvnunology 128(3):1458-1465, 1982. OtMsirrpport: National lnstitutes of Health and the March of Dimes. From the Departrnent of Pathology, Harvard Medical School, Boston. SPONTANEOUS T-CELL LYMPHOKINE PRODUCTION AND L•NIIAN('IiD MACROPHAGE Ia EXPRESSION AND TUMORICIDAL ACTIVff Y IN MRL-Ipr MICE Sekcted macrophage functions in MRL/Mp-Ipr/Ipr (MRI: Ipr) nuic were cvalu- atcd for this report. Speeifically, tluec macropluge furxtiuns were studied in MRI.-Ipr mice with autoimmurre lymphopruliferanve disease: surface cxpressrun of I-rcglun- as.sociatul (la) antigens, tumor eytotoxicity, and rntcrkukin-I (IL-I) pruJu:uun. MRL-Ipr rruce had a significantly increased rCpresenution uf la-posruve macruphagcs in thc pcntunc.l cavity. cumparcd to all normal strains of mice. In wdcr to study the basts of tlus u-casc. Wymocytcs or spknocytcs from MRL-Ipr mrce were trans- planted in(rapcntuneally into normal mice. Three days later the recipient mice had pcrituncal cxudates rx:h in la-positive macrophagcs. The cells which induced this response werz T cells which elaboratcd a lymphokinc responsible for the recruitment of la-positivc rnacrophages. In previous studies from this laboratory using mice, lyrnphokine was secreted only following the interaction of immune T cells with anti- gen. The resident macrophagcs of MRL-Ipr mice were activated and killed tunxx cells if triggercd by an interaction with bacterial producu, even without thc addition of lymphokinct. Secretion of IL-1 was normal. Results indicate that the drseascd MRL- Ilx mice arc characterized by (i) activated T cells that spontaneously secretc macro- phagc stimulatory moleculcs, and (ii) activated mxrophages that show both an increased expression of la and lymphokinc-indcpcndcnt triggering of tunrurxidal activity. Lu, C. Y. and Unanue. E. R. Clinical lmrnrawlogy and lmmunoparbology 25:213-222, 1982. Oncer support: National Institutes of Hcalth and the March of Dimes. From the Dcpanmcnt of Pathology, Harvard Medical School, Boston. V11. Epidemiology MORTALITY IN MIDDLE-AGED SMOKERS AND NONSMOKERS llic rclauun of cigarette smoking to orortality wu assessed in an 11-ycar follow- up stuJy of 4,004 nKn and women, 35-54 years of age, who mspurwkd to urging to havc multiphasic Iscalth checkups. Accounting fur 48 uther charactcnstics, both indi- vidually and in cuntbination, failed tu clinrinatc the association uf smoking with nxrn:rhty /rom all causes or with mortality from coronary heart disease. The sn>.rker- tu-rx,nsrnukcr nwnahly ratios, crudc and adjusted respectively, were 2.6.rrrd 2.114m all causes and 4.7 aixl 3.6 lur coronary hean disease. This analysis did not supprat tlw c(wntcrtryp.xhcsin drat thc association uf cigarette smoking with nwxtabty is accwrdary tu sunk underlying characteristic. Friedman. G. D.. O.rles. I.. G. and Ury. H. K. Thi New t:'nglun.! Juurrrul uJAfeJu•rne 3(70(5):213-217, 1979. UOther support: Kaucr Foundation Research Insututc. f-ruut thc Ikpanmc m of Medical Mcthods Rcxar.l, Kaucr-Pcnn.mcnt. M.dtcal Cuc I'rugram, Oakland, CA. Cn 00 T-4 T",( ~ H U .i 88 n9
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CIIAKACTERISTICS OF SMOKING•DISCORUAN'T MUNOZYC:OTIC '1'WINS Thc Kaiscr-Pernunentc Twin Rcgistry uf Oak)anJ. CA, cuotains uvcr 8,(l00 pain uf twins who volunteered their panicipatiun in a prugrarn uf ux:dical rcxarch on twins. In 1977 and 1978 a largc health yuestiunnairc was nuilcJ to all twtns agcJ IS years anJ over. After the fint nwiling in June 1977, a remindcr pustcard was sent in Octulxr 1977 to nonrespurxling same•scx twins. In this yuestiunnairc, each Iwtn was askcJ atxiut Itis on her own smoking habits and those of his or her coiwin. In the study presented hcre. Ihe smoking habits of the 33 cigarette snwkcrs in the snurking-Jisaxdant munucyguuc (MZ) female pairs wete compared with those of the 205 other MZ femalc cigarette srrwkers. Large and statistically significant di(fcrcrues were noted in somc mcasures of snx/king intcnsity. Ciguette smokcrs who had an MZ nrmsnruking crNwm tcndcJ tu stan smoking later and to smoke fewer cigarettes. This nuy cxplain, in pan. thc smalkrdrfference inCHDoccurrence between snxrken and nonsnxrkcn within snwk- ing-0ixordant twins than between smokca and nunsnwken in dx: gcncral population. Additional compansons were alsu rrurk between the drscordant snwkcn and their nonsmoking eotwins. Results of thesc comparisons show that, with regard to coronary heart disease (CHD) risk (actors, the discordant smokers were karx:r and consumed nwre alcohol than their nonsmoking cwwins. While thex Iraits arc ass.x:iatcJ with a lower risk of CHD, snwken also tendcd to be less educated anJ rcponcd less excrusc and concern about physical htness, consistent with higher rvsk. In conclusion, the data on a)imited numbcr of smoking-discordant femalc MZ twins suggest that, even with genetic ir1entuy, twins who differ in one characteristic may Jdfer in ut)x•r charactcris- tics rckvant to the outcome under consideration. Frirr4rxin, G. D. tl ol. , In: Gcdda. L.. Parisi, P., and Nancc, W. E. (eds. ): Tx•rn Rr.rrurrh f: F.)rrJnnrulugu ul nrrJCGnir'ul Srwlrts, New York: Alan K. Liss, Inc., 1'Ig1, pp. 17-22. OrArr suppwr: National 1(can, Lung and Bkxxl Institute. From the Dcpanmenl of Medical McthoJs Research and Ikpanmcnt of MeJicinc, Kaiser-Permancntc Medical Care Program. Oak)and; University of Cahfunua. LScrkc- ky; and the Department of Epidemiology and Intern:ruonal Hcalth, University of California, San Francisco. STRUCTURAL ANALYSIS OF SMOKING. ALCOHOI. US)i• ANI) PERSONAUTY FACTORS IN MZ AND DZ TWIN PAIR REI-ATIONSIiII'S This study presents an cxtcnsion of the testing uf twrn type cumparabildy tu a multivariate situation. Using data drawn from the Finnish Twin Culxrn as tlw: study populatiun, smoking chrvactcriuics, alcohul use patterns, and pcnunahty /'artora uf nwrxuygotic (MZ) and dizygotic (DZ) twin pain cuncord:rnt and JucurJant lur frc- yueru heavy alcohol use were described. The effcct of constraining cuvrriaruc rc)r- tinnships in individuals of either twin lype wys tested tu caimatc cuwp.rrabibty uf pairwise structures. Also, the cuv:rriance rclationshrps in individual twm. wcrc cuw- parcd tu thrne of singktuns. For such analyses, the general nxxkl lur thc cswnaurm uf Imcar strvrclural cyuation systcnu by nu trnxrm Iikchlxwxl un•Ilxa/s was .rpphoJ lo llk Finnish Twin Cuhun study Jau. Rewlts stx)wed that the Jrl/escn.cs tktwccn Ix:avy- 0 ~ anQ ligh/ drinkuig mJivrJuuls wcrc 111411,1 clear-cul On .inTlctum. Thcy JiIIcrcJ in the nkan surukinb Icvcls, and the czrr.rvcraun and neundici+m scures. (hc sanx: rc.ult ~ was obtained iu U'!, parrs discordant lirr Ixavy alcohol usv:. In dtxurJant M'L patrs, Ixrwcvcr• thc snxrkmg Jilfcretkcs were nhscrvcd but the personality I.ctur Jtllrrcnces wcre sccn in the ncuruucum.nd life Jusausfaction xalcs, suggcsung that the hasis of • JrscorJancc in ML twins may dilfcr from that in DZ pain arx/ sinblctuns. The paKxrr• X tion of JrxurJant pairs was 2996 in ML and 37% in DZ p.rirs, less than the cxpcclcJ 1-01 valuc. Multivariatc uulysis of dd(crcnccs in MZ and DZ Jiscurdant patrs con/irmrJ V the results (rurn thc univariatc analyses. the relative )rgnlhcancc of nCunNl6Unl in ML pairs Ixcuming wcakcr arxl statisucally nunsignificant. Smoking. ix:ur.dretvm. anJ )rIc ~ Jrs.atufactiun, inJrpcrxlcnt of gcnctic factors, sccrn to be indicators of pnac»es leading to heavy alcuhul usc. (rnginvainio, 11., Kapnu, 1., Kuskenvuo, M.. and Tarkkoncn• L. (Ranru%ulu, l.) Io: GcJJa, 1.., )'arui, 1'. and Njgxc, W. E. (cds.): Tuvn rrvrurc h 3: rprJrrmulugnus urul clrnirul sguJirs• New York: Alan K. Liss. Inc., 1981, pp. 23-35. Fruin the 6kp:utnx•nt of Public I/calth Science, Univenhy of Hebmkr• Hcl.rnkt. ('IGAKIiI•fti SMOKING, USE OF ALCOHOL. ANI) LEISUKIi-TIMI•. 1'II1'SICAI. A('1'IVffY AMONG SAML?-SEXED AUULT h1Al l:'TWINS 'llr rrlauun.hips uf cigarcuc smukrn);, alcolwl uvc alxl h•uurCdnn: ph)'ai.d a.'Irvity aurung:xlult mak twin paitictpanu in a Fmnish pulnrlatronn.tuJy arc )xc.cnt.J Ix•r.. For this cpxknuulugrcal stuJy, yuc.tiunnairc respixr.cs Irum 1•S i7 nxnwiygrMri (M! ) arxl i,5Q7 Jvygulrc ( UZ) nrale pairs agcd III and over werc analytcJ m tcnm uf cumbmuUuru uf th: three lactrxs and thc rclatrunship of these lactan lu each other ra rclatiun tu thc twin patr snuation. To Jrr tho• three facl.x analyses and an uvcra)1 c lu.tcr anaysis were carncJ out. Kcsults of thcsc Iests shnwcJ that rhe physical x'uvn) faidn nx:ans were alnwst cr>rtstanl with agc- but there was a dccrcax wrth :rgc in alcotol c1NNun11Kxn1; Irlr tIh' sm.lking faii:1rM, there was a steady Increaw with dl;e until SO-u ycar.,aftcrwhichasli(;htrkcrcaxtwcurrcJ Thccurrclatrunnwlhcrciu.tktwc.ndk Lrctun in the wlx.IC Xnc.% shUwCd a high cuRClatxNl between cll!drClti .nxrkmg and u>e uf a)c.rlxrl. rnd smrll negative cunaati.ms lur phy.rc.A aaivuy JIxl crg.rent .mukmg aixl lor physrcal :x'tivuy and ux ol akutxd. In drw clustcr .mjlya.• cight cluacrs wcrc LrunJ to hc aabk in l:n)up-rc);nwp.itu.ruun. Whhuvcr'A/' ; ul nx•nrh.•r. rcuoarnmg ur tlx•..mx: clu.•cr Irom on. analy.u tu an.Khcr. AN xo twm.hip. txah M/. anJ 1)Z twin parr wcmt.crN wrrrc in thc.amc cluacr much nxnc .dtcu dwu.vlvilc.ln but t1x M'L-lyl./lrcra)I JIIL•rcncc Was rclallv.I)' sIIW)I. Thk' hIglK'l ML. I)/. r.ruu. ul ..hxrvcJtoc.I~.itrJilua.nnEratc.wcrcmtWUawtcn'A/:lu.tcrn,~ 7•x)INhh.N) hrvros wuh .r hqh ux•an dcgrcc u/ Icrsurc-unx• )rhy.r:.tl xnvuy; aixl l31 dk v.r)'.nuII clu.rc( nu. N, wlurh had a very hrEh uw.rn r).ulxr) u... K rpnu, 1.. Kuskcnvuu, A1 Jrxl S.&tnW. S. (HunrowL.. l, 1 In: G: dJa. I-.. I'rnm. I' .rnJ N.rnr.. W. ):. trJ. l. Tu rn i.•sr,rr, li (.l..J. rru.•l,.crr.rl ,ui,l rbru„d %ruJr,•,. N.•w 1'.uk: Al.rn K I.i... Inc. • I`/141- pp 1)-J7S I•rt ui tlxt I )LpJrtlucrd ul I'ublic Ilrallh Scl.rx. • l/nivct.ul ul Il:lanki, Il:lauki 'xl vl .
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SLELP 1)ISOKD1iRS IN KGl•ATIC)N TO ('OKONAKI' IIGAKI• 1)lSliASI: GviJcn.c frunr an Amcrrcan study indicating a rclruun.hip Ixtwccn .Iccp timr and nrsrtahty, including death frunr curunary bean dtxas. (CIIU). led to a F'innt.h Twin Cohurt study on the relationship between slccp time :fnJ CI If). In O>` cpiJcmiu- logical paper presented hcre, lhe skeping time Jistnbuliuns in the U.S.A. taken frum the Kripke study werc cwnpued to data obtaincd in 1975 Gunl 5,419 Finnish adult mcn, and age-standanfized prvpoctions wcrc computed fur n>ln ay;cd 30 and over. Ke.ult% showed, f)rst of all, thal the proportion of mcn skcping 9-1O hours or nuxc is higher tn Finland than in the United States. They also showed that shurt (Ics than six hau(%) or lung (nrur+e than 10 hours) sleepers had signilicantly nwrc cunfplatnts relating u) CI11) (han those who slept for sevcn-eight hours per night. ShurfeneJ skcp was cspccrally related to angina pectoris and pain of possible infarctiun, and currdated with abing, ptwrr sleep quality )urd/or Type A behavior pattern score. Long slccp. on thc ulhcr harxl, was corrclated with good subjcctive slccp yuality, but this gr-oup had tl>` highest irxidcrrce oldiagrrosed myocardial infarction. This relattunship held alter sututically controlling for many possibk confuunders such as hypcrtensron. Jrug anJ alcohol u+c, snsuking arrd Type A behavior pattcrn. In additiun, Ilu cardiuvascular physiulugy and pathuphysiu4rgy of slcep is rcvicwcd hcre ux) thc rclauunship of surnc s{xcrlic slecp dixxdcrs to CHD is discusscd. Partinen. M. e( ul. (Runrosa/u, 1.) Ar•lu MsJrra Scaadifwvira (Suppl) G60:G9•10, 1982. Other support: Finnish Foundation fur Cardiovascular Kcx:arch. Fra>rrr the Departfnenls of Neurology. Physiology and Public licalth Scicncc, Unrvcr- sity of Helsinki, Helsinki. MULTIVAKIATI: L(X;IT ANAI•YSIS OF CONCOKUANIG KA Ill)S I OK QUALITATIVE TRAITS IN TWIN STUI)II:S This statistical paper prcatnts a new approach fur tlk anrlysts ul a icltaln tyIw rt/ twin dua. The rtwrkl that is applicd in this paper, the lugn Ina><Jcl, u analugou. tu the widely useJ log-hrrear model for conungency table anrlysi.. Thi+ nxxkl, whtch prr• frrits testing of interacliw) effects before eslinuting the rnain ellects uf the study variables, may also be easily extendcd to (our-way or even nrnc cumplcx lablc., though the testing pnleedures and seyuential hypaahesis testing txcunxs incrcasiny:ly dcfrwKling. In Ihe examplc uscd fur this stualy, data Irufn a cro~.-urUunrl audy of cigarctte sawking anwng adult twin pairs in two cuuntnc. w~. uscd. The nwluvrnrlc aasessrnent of genetic fachxs in relation to uttx:r facatrs was carried uul hy luvu analysis of cuncrxdancc ratios by analyzing three varrablc.. zygu.rry, xx, and cuun- try, at thc safnc tinre. Thus, the effect of scx and cuunny un tygu.ny tn th. .mu{,uiti uait cuuld bc contrull:d. For ciguctac sntukrng, tlx pro.I nl results mJtcrtc that of t>, ldt countries the zygosity effect is signdicant, rnJ ifuklxn.knl ul l'Iwrrtfy :I/IJ .rx. Al- though not presented hcre, the resull.l>LW (arr buth curr.nt rnJ ever cigarru.• .nnrltnl; as well as for arrwking uvcr urx pacl a day crdkr curr.ntly ur ever 1•uf th. Ir..tvy snwkcrs, thc zygusity eifcct haJ a stgnnccrnt Interaction with scx. A.a):wb:rut ity effect nnplleG greater cOnCnrdalKe fUr the trait arrNmg MI thJll I)I prlrsL/IJ I% aIUC (u the irfentical gcrarne anJ probably greater cunm>,ar cnvuunatcut ul b1L twill p,ur.. Kaprio. 1., Sanra, S.. arx) Kuskcnvuu, M. (Runru.tuh.. 1.1 Artu Grnrnrur rf Mrdicur GrmrUuLigiur .W:2b7•274, 1941. Frum the Ikpartnrcnt uf Public NCrlth St ICIKe, U/rlvl'rNty UI IIII,lllll. IIiI,11111 Active Projects Following is a list of Q)c principal investigators, or institutiorts, o( projects under way or activated in the period since the previous Report, togcthcr with the respective project titles. Completed projects are listed in a later sectioo. PRINCIPAL INVFS77GATOR OR INSTITUTION JOIIN 1. ALIiL'KS, Pul)., Rrxurrh Aa- ~.H'inlr l'ru/ruur u/ Alydi(inr. Univcr- Suy uf Wasbinstun Schuol of McJicinc, Jcattlc. PKOJEC'T T1TLE Ili{h aknmily lipupfutdn yuanlhation I/AKKY N. ANfI)NIAUIS. 1'rr.l), ('r..- /r..,,r ,./ (I.,.,l,r.rwur, Ilr(varJ Uni• vcrsny Schoul uf Public Ftcalrh, Buslun. -r11c)nfns xt. AuNI:. PnD., ,rJinndl rn J.nunrn.'/ut'Y. 'fhe Jewish lluspilal u( St. Luuu. UI:KNAKD M. IlAB1OK, M.D.. Pu.D.. Pru/ri...r u/ A(rJ.rrnr, New IinrlanJ McJtc:d Cenrcr Huspnal, Hustun. LIiS111i nAliK, A11), Arrunarr Pnr/er• ,ur u( A1rJirinr, ('olumbu Univcrsny ('ullcJc of I'hysicrans k SurJcuns, New Yurt, 111'A 1. van Jcn 111:K(:, M.D., RrneurcA PcJwu-n.'iun, AJlumr Pru/rti..r in Nio- sf.mJlGrt. Umvcnity of Cali(urnia School of Public Ilcaltb, Oakland. KI(-IIAKD 1. UING, M.D.. Pro/rs.ur n/ A/rJ" rmr (rmrnnnl, (lruvervity of 'uuthcan Caldut ma Sch.nrl uf McJicme. Lus An6a•Ih: I'n~rni.~ A...nwUr rn lf..n orrJu.d 1:rnl;urr.•rny;- Cahfurnir In•In• ulc uf 'Icihnulu/;y: (rrrr'. rw ../ CurJad. „x v unJ Jnn.un.u.J AlrJi. rnr, HrmtinF. ta.n Alcnuxirl Iluspual, 1'as:rdcna, C'A. 1)1 IJA/t( 1:. 111SWAS, IhrD., D 1c.. ../ UruJ /4ub.A•c, I rliuratury of I'harmauduLy, Ilrrvard Scboul of Uc111.1I Medicine, Bustun. IKA u IrI A(•K, At l)., Pru/r,a.r „nJ (•lu,-:. Ihri.Lnr „/ 1).•,iL.Jnnavuul N.•rr .../..gr• Curn.•11 Untversny MeJr:rl C-ul- IrA;c, New l•urL. I'IIl 1 1 1\ It III .1IK• 1'it D. r ../ l.n ...6.+r, lhuv.•r.u)' u( CrlJurn,... Itcrlclcy• Flunun rlatcict-JcrrvcJ srowlh factor (PDGF): relauunshrp ro human athcrusckrusb lmcr(crun-aaarvaliun of supp(eswr T ccll pathw,.ys StuJies on the mechrmsnr of activation of Ihc respiratory burst in ncutruphds Ci{arcuc smoling in nurmurrnvivc and hypcrtensive subjects b1uuJ pressure, renin, aldusrtrune and catechulamine responscs Pulmonary function tests in adolescents and their parents-an eprJemiologie appruacb Chulcstcr.>r inhibition and carbun monox- tJe and arbcrusclcruau l.ipoprutcins rnJ the artc6ial wall E/lcc(s of nicotine and bcnzu(o)p)rcne .m hurmunc prwlucuun Nicoane.nJ ncurunrl Jcvcluptucnt Kegulabun o( natural LJlcr ccll act.vny 94 95
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CORONARY-PKONE EiEHAV1OR IN ADULT SAME-SI:XEU MAE.E TWINS: AN EPIDEMIOLOGICAL STUDY En this atlcmpt to idcntify farnilial and cnvironmental cunyxmcnts of coronary- prvne behavior paueau, the rcgpoasu from 5.419 male twin pain in the Finnish Twin Cohort to a 1975 questiawirc were investigatcd in several differcnt ways. To begin with, thc postal qucstionwirc study provided data on zygosity, smoking, alcohol usc, kisurc-timc physical nclivity, weight, hcight, and drvg usage. Psychosocial factors such as muital statw, occupation aod occupational history, changes of rcsidence and employment, extroversioo and lability, .nd type A behavior wcre also studicd, as wcll as various symptoms arad history of disease. Type A behavior pattern was measured by the rating scale developed by 8artncr. Rcsuhs of this study showed that the intraclass corrclations were 0.251 for rrwnozygotic (MZ) pairs and 0.052 for dizygotic (DZ) pairs. The heriubility estimates were higher in younger than in older age groups, arK1 the proportion of A-type concordant pairs also showed an age aend. Whilc the prolxu- tioo of MZ pairs arnong A-type concordant pairs was grcater than anwng 11-type concordant pairs, U+e difference was rwt statistically significant. In this study, an association in men betwcen A-type behavior pattern and angina pecwns on the Rose questionruirc was found. Morcover, the discordant pair analysis Ixe.untctl hcrc abowed that there were some envirwrmental factors clearly associated with coronary- pnonc behavior. As of now, A-type behavior has been shown to be an independent rnk factor for different manifestations of coronary heart disease (CHD). Since surne psy- ehosocial factors, such as marital status and social class, which were found to correlate with A-type behavior in this sttrdy, are known to be associated with CI ID in Finland, it seems reasonable that the relationship of A-type behavior, psychosocial facturs, And CHD should be investigated funher. Koskcnvuo, M., Kaprio, J., Langinvainio, I(., Rumo, M. And Suna, S. (Rantasab, 1.) In: Gedda, L., Paisi, P. arNl Nance, W. E. (cds.): Twin rr.rrun h.f: qnJrnuuluArru! and r'linirul srudiei, New York: Alan K. Lus, Inc., Nlltl, pp 1)'1-14N. From the Dcparunent of Public 1(ealth Scierxc. University of }Iclsutii, liclstnki. FINNISH TWINS REARED APART: PRELIMINARY CHARACTERIZATION OF REARING ENVIRONMENT This paper prcscats some characteristics of the rcaring cnvirumnent of 478 FFrn- nish-speaking, adult, like-sexed twin pairs raised apart from the age of IU or kss. Ti>c Finnish Twin Cohott Study provided the raw data base (or this study, and twrnshqp was confirmcd by a questionnaire study in 1975 that cuvcred health-rclatcd rtcros And standardizcd measures of rttorbidity. In additiim to these qucstiuns, a number of othcr aspects were considered: whether the twin pair lived together and. tf nut, at what agc separatioa had occurred. The present frequency of intrapair cuntact, birth urdcr, And handedness were also investigated and questions directed to zygosity asse»nx:nt wcre included. Later, during November 1979- January 1980, a new queuionnauc un Ihcir childhood cnvirwunent went out to the 478 twin pairs in thc lcst group atrl thrcc correspooding corwol groups, which were formcd to asscss which .apccts uf tlx rearing envuonmcru, personality factors, and childhood medical history of tlk study sampk differed (rum those of twins of the same age And sex. Listings fur thrs study arc given by agc ul scparauun (groups I-IV) uxl by birth year and sex. Results of this study showed tlut the mtrapair corrclatuun of rearing cnvironmcnt vaned grcatly as it ap- pearcd Irum vuubks such as age at scparation, family rncmbcn, schuol, friends. living place. intrapair contact frcqucncy, and education and occupaurMt of rearing parcnts. Morcuver, the cause of scpuation, based on scl(-repcM, seemed to bc fairly often associated with some psychusocial pathology. The separation of members of a twin pair may also mean intrapair selection. Further assessmcnt and comparisons with singletons from the general population and with psychiatric outpatients arc ongoing for this study. V Langinvainio, I1. et a!. (Rantasalo, 1.) ~ In: Gakla, L.. Parisi. P. and Nance, W. E. (eds.): Twin rtsrarch 3: intrllrgtnr•c, pcrsonaliry, and Jtvtlupmcnr, New York: Alan R. Liss, Inc., 1981, pp. 189-198. Fmm Ilte Ikpartrnenls of Public Ilealth Scrence arxl Psycluatry. Umvcrsdy of Ilclsinki, Ilclsinki. CANCER IN ADULT SAME-SEXED TWINS: A HISTORICAL COHORT STUUY In this altcmlx lu investigate the feasibifity of utilizing the twin method as a case- cuntrul type of study, a historical recurd-Irnkage cohon study between the Finnish Twin Coh.m Study And the Finnish Cancer Registry was earricd out. The Finnish Twin C'.rMin was crcatcd in 1974 from the computcrized Ccntral Population Registry, while thc Finntsh CuKCr Registry is A pupulation-bascd, national rcgi.try rn opcratnxt since 1451. It is cun.itkrcd tu trc ratfkr compkte with respcct to incitknt cases of canccr in fmlan.I. fur thu study, pcrstms included in Ixnh regisures were idenutlcd by cum- p.rnn); by cumpurcr the Ikrxrnal irkntilicatiun nurnhers (a 10-drgtt umque .t>a1c as- st);ikd tu each resrdcnt in Finland) of the twu registries. The companaun cuvered the ycus 1'Kr7-1976. 'Iltc twtn recurd linkage yicldcJ thc observed nuntbers of cancers uf ddlcrcnt types. Age And sex-specncc pcrson-ycars at risk were calculatcd scpuatcly for the twin pupulatirm and the singleton group. Also, pcrsun-ycan at n.k wcre calculated for cruwms of cancer probands. Results of this study stwwcd thu the total carkcr morbidity rn the twin population was lower than expccted for buth men and women. The rclative risk for all cancers was 0.77 for men atr10.72 fur wumen. In the singlcton pupulatKm, the relative risk for men was slightly ovcr unmty. In this study, the rauu of the obscrved to expccted cancer morbidity closely reflected the saandudtzcd (tutaq nwnalhy ratios fur the same calendar years, suggesting that the luwcr-than- ex)xctcd cancer mrxbrdny may have a backgrourrl in common with the luwer-th:ut- cxpectcd mortality. Also, the low concurdance rate found in this sttuly suggc.ts that n uuy be fruitful to study the environrocntal expusure of cancer-discordant MZ and UZ twms. Kaprru. J. e-t ul. (Runtusulu, 1.) In: (icdJa. L., I'arui. P. And N,ur.•c, W. Ei. tcds,): Tuvn rr•reun h J rprJnn~,.luKrual unJrlrnrrul xtuJre.r, Ncw York: Alan R Liss. Inc.. 1981. pp. :17-2_'.1 fruut tlk Drp.rtmcnr of Public Hcalth Sirencc, Umvenity of Elchwkt, atal I-mrnuh Cancer Registry, slclstnkt. 93 92
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PRINCIPAL INV!$IIGATOR OR INSiTCU17ON J. MARK BRAUGIILER, A...u- PROJk;CI '1TI7,>r. '1 be altcraliun of guanylalc cyclase by tanr Pro/esrur oI Pharnracolugy, Norlhcaslcrn Ohio Univcrsitics College of Mcdicinc, Ruolstown. EDWARD BKESNICK, Pu.D., Prultuur and Cbairnrun, Dep.utnunt oj Biu- chunisrry, Tbe Univcrsrly of Vermont CoUege of Medicine, Burlinjton. REBECCA BRYSON. Pu.D., A,s.x•iare Profcssor u/ flyclwluYy, San Diego S1ate University. San Diego, CA. ndruc oaide Eaprc» iun of cyluchlonsc P43/>< Inleraclive el(ecls of nicutine. Iestuvlerone and estradrul on wcrght change, fu.Nl cunsumplion and aclivity of malc anJ lcmalc rals under high and low pru• Idn dicts VINCENZO BUONASSISI, M.D.. As,..- ciurr Resrarch Biuluyi,r, The Univer- sity ot California at San Dicgo, La Jolla. DAVID L. BUSBEE, Pu.D.. Duecror, Grnrrirs Centn; A,s.xrurr Pru/rssor u/ Biuloricul Scirncrs, Norlh Tcaas Stalc Univcrsity, Dcnton. EDWAKD 1. CAMPBELI., M.D.. At- sumnr PruJr,swuf Mrdn(nr, Washing- lon Univcrsily Scbool of Mcdicinc, St. Louis. WII LIAM A. CARTER. M.D., Pru/c.,ur ..1 llr.nund..Yy and Alcdrral Onculur; y. Ilahncmann McJiul College. Plnla- Jclphra. ALBEK'r CASTKO, Dirrctor, Jlw- nrunr Rr,rurrA LaAurutwy; Pio/r,sur ol Parbuluyy and Aledicinr. University of Miami School of Medicine. Miami, FL. FRANCIS C. CIIAO, M.D.. Pn.D., Sen- ior lnvestigator, Center for Blood Re- iearCh, BostoO. JAN F. (-111 LBOWSKI, Pru•D., A„i,mnr Pru/nw.r ul Brw hcr.u.suy, McJical ('u1- lcgc of Virginia. Richmond. CURT I. CIVIN, M.D., A...i.runr Prn/rr ,ur u/ Unr.duKy 6 Prdwrrics, lhc Jobm Hopkins Oncolugy Centcr, Balu• more. CHARI.FS G. COCHRANE, M.D. A1nn• brr, Dcparrnrcnr oJ LmmrunoparAoloYy, Scripps Clinic and Research Founda- tiun, La Jolla. CA. lleparan srd(are proteuglycans and blurnl hunu:uslalrc mcchanurns The bin.hcmrcal anJ physiological char- PRINCIPAL INVEST7GATOR OR INS-TTIUTION BIiKNiI'1i 11. ('O111?N, Ihr.D.. L'rnJr,.ur w.J Dlrr.rur, lJruuun Grnruu/Grnrnr 1(pr.b'nuuluKy Pr..~nun,'(he Johns )tup- Lrns Unrvcrsny School of Hypcne and Public Ucallh, r(allimure. KOBFK'1 W. (Y)l MAN, M.D., Pru/rrwr u/ AJr,lnurr, 'I'cntplc University School uf Medicine, Philadelphia. GIUON ('ZAI'\KI, M ,..r u/ Pl.y,nul ('Arn.nuy, lhc 11cbr,cw (luiveraly, Icrusalem, Israel, KUIII;KT f•('111. I•rr 1). Pru/r,rur uf Ana- runrv. Mrclugan Slalc Unlvcrsny, lias( Lansing. CAKLTUN K FKICKION, Pu D.. Pro- /r,,.n „/ Phun,,.rul.,xy.-lhe Univcrslty u( Teaas College of Pbatmaey, Austin. PKOJECr 77TL[ V. Q Gcuctic-cpidcmrulugrc characlcristics of T snwLcrsanJnonsmulcrs T Initiatiun of plasma coagulation anJ kinin fornring systcros in man ~ \ On Ihe lusieity of usygen anJ superoa.de a ion: is wperosiJe luaic? The e0ecls of hypuaic byposu• carbon uuuuarJe anJ lrealments rn/fuencing hypusra tuaicny on enJucnne-bke cells in auways u( young rabbits and rabbit fctuscs BIuuJ-hrain munuormg of sustained nico- line levels in rats aclcristres of a prutcin which spccih- cally hinds polycyclic aruwnanc bydru• V. carbuns Modulalors of m0ammalory ccll protculy- lK aClisnly A The inlcrplay u( mrmunu.uivcillancc anJ Intel(erun InJucllun /11 tlmNnlgene\1s (:HNIi (iK1VIN. Pu.D.. Pru/r..ar a/ I'b.ornuru(uA•y; L)rmr• Univcrnoy of Colorado Schoul of Pharrnacy, Boulder. VAN K. FI INSIFIN• M.D., Pru/e..,.r ol Alydu n.r d f p.J.•..uulury, Yale Unr vcruty Schu.d of Medicine. New Ilavcn, ('1. Acliuns of nrcuunc on rsulalcJ pcrfuud mouse brain Ellecls o( nicotine on neurupepnJe secree- Iwn by intacl muusc brain, a pharmaco- gcnclre study SmoGng. Jclcctiun bras and prru.ary lung canccr Jl).M I'll 1). 1•1 l l)AIAN, As,l). A.nuu.n..- L•I(ccls o( agutg and Lprds un d.c rmmunc Nicutinc in bl.xa<I: JcacAiun by raJtu• p.uhalnq•..r, 1a..p. ('hnK anJ Kcscalch 1 uundauun, l.a ~o11a. CA. syslenl imnl4461u4.,ay l l It)A)A1 at. 1•INI.AY. I'n 1).. .(...a uur Slruclure, prupenccs and regulaliun of 1'ru/raur u/ Ol•,rrrnr. un.l (: vnr. uluFy. Ncw 1'urL Unrvcraly Medical Center. nurusc plavna prutcasc mhrbuurs New Yurl. Platclet activatiun and blood hypercuapr- labrhty UIK(:11-I A PI.ODEKUS-MYKK(II D, EpiJew.ulugic tesearcb on the Swedtsh I'n D.. A„urunr Pra./nr.x ../ f.nvuan- twin rcgistrrc, rnr.uut LLyArrne, The ICarulinsla Insu• ' lutc Sloclhulnt. alufimcln. rnvcaiF:.uuu u( prutadn,.x'-1 C , maa ugl.dwhn mtcr acliun JUl)I'fll ANN I't)SI'1-K. Invulvculcn/ of claa.n rn lung J.scavc ..,r und C'l.mrprn.ur. l)rlprrr.nrnr ../ 14..luy,v, Syuacusc Unlvcrsny, Syracuac, Bluchcunary an.l functiun of huroan gtin• NY. ulupurclrc anliicns JACK W I K.1N).1 I, 1•n 1). ('..uudr,uu SmoGnr; .rnJ luag ...nccr Jugn.ulrC lesl Medialiun systcros in nJlaurnralury Iung disease u. AlrJa ul H: .:..ro b. Vcrcr..ns AJnunrs• Iralrou McJtc.l ('cmcr. Bay Pines, 1-L. AII.AN P. FKIiEDMAN, M.D., A„u- rmu Pr../rsrur ../ AMdirrnc, Hahne• mann Medical Collegc, Philadclpbra. lu rJcnufy pcrsuns at high r»k 71sc cllccl of cigarette snwlmg on Ibe alveolar clcarance ralc of Incrt dust paruclcs in the human lung 96 97
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PWNCIrAL INVk$T7GATOR PKOJI•:('1' 'l7'I'LK rR1N('IrAL INVI•'.S17GATOK rxOJECr nlu ~ OR INSI'ITUI7ON UR INSCCIUTION Pu.D.. Xulfl Sri- FKEFAIAN AAKON li RuJcnl and human lunY cpllhclnl cell cul- NUII(1YO111I 11AGINO, A1.1y.. 1'/l I).. NlcWinlc rcccplurN uf I I1K11 a.un tcrnr ~ • . ('ahfurnia UirxncJical Kcacarch .vuur ture as a tool fur caranuAcnesu rescarch Pr..J.•...,r .,/ .Jnur.nuy, Univcl.ny ul ' inals In the Incdian cmincnce , FrwnJaliun. La Jolla. CA. in vlrru cnter, S.n An• Tcta• Ilcallh Jclcncc ( ~ KJFI 1. FUX1:• AI.D., Pn,/r,.l.,r.,/ Jliu,.l• .,gr• 'l/x I:rrulinska Inuitulc, SIUi1- hulm. M(tK1UN GAl USfON. A1.U., A,,,,ruur Pru/r„ar .,/ AlrJlrinr, New 1'uik Unr vaury MeJleal Cenur, New York. Af1CflAtiL C. G1?OKAS, M.D.. Pru/risw and Vire-ChaKman, Drpool• rnrnr u/ AlrJiainr. Univcrsity of Cali- fornia School of Afedicine, Davis. TERESA GESSNEK, Pr(.D., Arsociarc Cancer Rrsrw:h Sclrnli,r, Heallb Ke- search• Ine., Roswell Park Divuwn, Uu/lalo. JACQUES E. GIELEN. Prr.D.. As,.niurr Pru/rsjw, Labururory ../ AlrJirul Cl.cnlurry, Tori:olory and Ilygienr• Inuilule uf Palbology. University of Liege. LicSe- UclCium. C:AURIha. C. GODMAN• A1.D.. Pr.,/r,- ,ur .,/ P.uh..L,l;y, Columbia Univcl.ily College of Physicians 4 Suricons, New York. WARKI:N A/. GOLD, M.D.. Pru/n,ur u/ AlrJra•uu• CarJiuvascular Kcscalch Institurc, University of California, San }'rancirco. SIDNEY GOI.UFISCHF.R, ALD.• Pru/r,- sw u/ Patlu.la.y- Albert tinuein Cul- /cYc of Medicine. Tbc Urona, NY. htAUKICG GKtEN, At.U.• Dircrrw, br ,luurr /..r Aluf.•rulw VuuluYy, St. Luuu University Medical Center, St. Luuis. A1AK1: 1. GRfENE, Prr.D., Ai..xlurr Pr.,/rawr u/ PudluluLy. HarvarJ McJ- ical School. Uouoo. ii1KA L. G11K1()n, D.V.AI., M.V. Sc.• A,IU'wrr Cuncrr Rrxw.ll Sclrnlur, Drpurr,nrrrr o/ E.prrirnerl/al Tlmruprunu, Rosweil Park Memurial lnu/tutc, tlutfalo. f pilhclull ccll carcinuy(cncsia u/ rvuu Niculiue, i.llcihulamines •anJ nrulrhndu• cnnc funcilons .Uia><lrcmical ba,iN of plcdupualiun tu cluunl'; ubstrucuve pulmuuary Jncasc C'uculaling panclcali: claslaac 2 anJ cm- ph)xma In man Pbarmaco`enelics of eonjugations and lung canccr risk AluJulanon uf aryl tr)'Jlucarbun h)'Jrusy- lase unJ epusiJe hydralase in aninul ti»ucs anJ irl ccll culthue by cii;areuc unuke cunJenwlc and rnhcr chcmicals Uiucbcmical lucchani.un(s) and qu'alila- llve and quanlilalive cunxquences of bcnw(a)pylcl>< mclatwdi.m C•ylu.ldclal ulFanirauun u( tba• cnJo Ihclial ccll in Ic);ulalwn ul .h-Ipa' cun- Iracuhly anJ aW IaCC nwvclucnl Li1Tcc1 of uiune un airway masl cells Lauaccllular rnalris-cylrxhcnlntry and ullraslruclure Anlpllhcatrun of hunlan aJcnavinn tran, formatron pnncios In p/ularpul/c and cukaryulic cclls Supp/cswr cclls in syn);cnclc luluur iln• mundy Kulc ul gcnct/cs :InJ p.d)vruuutlc byJlu- cartwn ulctal,ulranl /n human lung can- ccr CAKOLINIi 11 I IAI 1, M D• A lluriulr 1'n,/r.wr u/ I'n6.urn, wIJ Alyd,rlnr, (lurvenily of K.o,hc.lcr Schuol of McJ• /:uK. Kuchcacr• NY. I INUA AI. I/AI 1, 1'u.U.. A.%.nu,lr 1'n,. /r..or u/ (irnrnl, unJ Nrurnuvrnlr. Alherl lim+lein ('ullcge uf MeJl.ine uf 1'cshrva Umvcl.uy-'Ihc Urunt, NY. I'AUI. IIAMU\I1. M D.• Ar,.,.,ulr l'ru- /e,wr u/ Phy,iulury and hwplquc,, anJ AfrJl.ulr• Georgetown Univcnity Schuoh uf MrJlcinc and Ucnluuy, Wash/nYlon, D.C. NUKMAN W. IIIi1MSI KA, 1'lt D.. Pru- Irsrw u/ P,yl//..l.,ly: 1)Irn'lur, llun,un Farrurs Lahurulury, Univcr.rly of South Dalula• Vanulhun. 1f1:KUlK r I). 111.KS(:OWI'r%• Pll D.. ,4,rucwre Pru/r..,ur u/ Ahcrubiuluw, Gcurgcwwn Umveruly Schuuls of McJ• icine anJ Denli.uy, Wasbinslun, D C. KOIrPKT hl. UO1 i-MAN, 1'u.D., As- .ulunl .,/ Pr,liurnc, ul Rru- Jrn,r• Unrvcr.ny ••f Califurnra Sclarul uf A1c.Dculc• I a lulla. 11 KOMI 1. II(UNA('1:1. 1'u.l). .1..,.• (huvcr.u) ul I uwcll, I uwcll. AIA. WAl'Nli 11(1\l, Ih1.D.. .(.uh,urr /r...w. ('culcr l.u I(lain Kc.calih, Uuivcl.il)' ul K.wlrc.lcr McJical Ccn- ler. KuKhlatcr. NY. AAKON JANOFF• Pu D., Pru/r„w oJ Purbulul;y, Ilcahh Scienccs Centcr- Slate Univasuy of New York at Stony Uruuk, S/ony Utuuk. MUKKII I KAKNOV1K1'. M It.. If. (ll.- .)b.ulu, L l'r../,..,'r „/ J'.ula.l,a•,...1 .Inu- n,ury, ItarvarJ M.J/ia1lcM,u1• Uuslun. IN(.1 <.:\I<1) A1 i.l l I 11. I'u 1).. .a..l.uu,l I',,./r..,.r „I .Inul,.un'. (bura'nn) uf Wncun.m l.iuwl uf Vclclln~ly A1.J1• .ulc• AI•IJ/s„n. ~ U rn Ur.nuplulu n,rlunu);,nlrr slr.ins ~ ('/f.ucu. .,nu4c anJ hp.•p/ulc/n IcnnHkl• InY by Ihc hlnr Sunlc hchavioral a.pcch nf amu/.ins and snwkmg Jcprivauun tafeils uf ci4arellc +uwkc a•spu.ulc on do- vclaq>rncnlal. cellul.u anJ mo/r:ulal a.• pccl. uf thc luuunrlc Ic,pun.c Mclhiuninc J:Iwn.l.•nic. mclll)l.u,un and u1 sa11rC II,/rNl.n Iu..Uuu Ka•~ulauuu ul acllul•u una•.gina•. NKWI/N'~In.lu.cJ chanYc, ul plim.dc lu{:h Jcmu)• Ilp.q>ruwcm, YluJrc, uf m.ulmc /nlaa.uun wuh b1.wJ cclls Furthcr ,tuJn•% uu ,upplc..wn ul pru• leasc InAibinwn hy ugalcltc )/uuke Imnlun.doyi. a»ay uf hutY aaslin Jc- YI aJatrun fha• luukcular haa,ul pldolanal) .u11ai- lam .ccrcUUU Ly 1)/w I1 I>.w'uun..)la.. uuJlca In /ntxt .cU, .nJ a .c11 /rcc syucol ILrI 1: 1 ung ncuruan./.wu/rc arll mncrva- Iwn InlcrrcLuiun.hip uI In/cUluu. luwcr rcv (~unlu/y Ir.wt Jlvvv /n ,n(.,ncy and lunl anJ cnvuunnw'ul.,l L.al.u. to laler .kvclupluelu of ahlum. 111411: Jneax Gcncuc Jilfcrcnces in niculine .cnailivily Pal1 11lrun.pla.cmal :II:a ..I ruuLmg un lung IKUfUa'IIJ.K1u1N ccll. in the /hWiale 96 Y9
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PRINCIPAL INVFS'E7GATOR OR INSEITUTION KOBERT W. KKEILICK. Pu.D., Pru/rs- sor o/ C'hrnlisrry. Utuversity of Rochcs- ler, Rocbcstcr, NY. F(LAUS E. KU1=TTNEK, 1'1/.D., Pr../es- sur unJ Clwirrnan; Drpartnrent u/ Bio- chrrrrisrry, Rush College of Ilcallh Sci- enccs and Rush Mcdical College. Kusb- Presbylcrian-Sl. Lukt i Medical Centcr, Cbicago. LAWRE?NCE L. KUPPER, Pu.D.. Assu- riurc ProJrn..r uf Biusrarissics. Univcr- sily of Nor/h Carulina School of Pub- lic Eicaltb, Chapel Ilill. ADEL LA)THA, Prr.D., Dirrclor, New York Statc Rcscarcb Institute for Ncu- rocbemistry atsd Drug AJdiction, Plew York. DON LAPENAS. M.D., Assislanr Pro/es- sor o! Putllulupy. University of Vcr- mont College of Mcdicine, Burlington. E. CLINTON LAWRENCE, M.D., Assis- tunr Pru/esror of Mcdicint, Baylor Col- Icgc of Mcdicine, liouston. PHILIP M. LE QUESNE. PII.D., D.Sc.. Pru/essor ol Chemistry, Nurtbeastern University, Boston. FABIAN 1. LIONETT7, Pe.D., Research ProJeuor o/ Blochemisrry, Boston Uni- versity School of Mcdicioc, Boston. GESINA L. LONGENECKER. Pr/.D.. Associure Pro/rssor o! Pharetacufugy, University of South Alabama College of Mcdiciac, Mobile. RONALD B. LUFTIG. Pu.D., Pru/es,ur ol Aficrobiulogy and /mnlarnuluYy. Utu- vcrsity of Soutb Carolina School of Medicia, Columbia. HENRY T. LYNCH. M.D., Pru/rssur and Chairnran. Drpartmenr o/ Prevrn- rirt Alydicinc and Prrblic Hralrh, Crcighton University Scbool of Mcdi- cine. Omaha. BRUCE A. MACHER. Pu.D., A.r.isranr Pru/rssw aJ PJwrnlacrurlcal Cllernistry. Universily of California, San Francisco. ALAN C. MtLAUGHLIN, Pu.D.. B/u- physrcur, Brookbavca National Labora- tory, Uptoq NY. rKOJk:C 1' T1TLE lnvcsugauum of t11c mlcractiun of nicu- Gnc wilb urcrrrbrancs Local rcgulaliun of nurmal and patbulugic invasion Kcgulalion uf prulifcrabun of invaaivc cclls VcrdicaUUn u( a stali.tical agc-pcriuJ- cuhurt analys/s of lung canccr rRiNCIrAE. FNVPSE7GATOR OR 1NSI7T'UT1ON l. WISTEK MF.IGI, M.D.. ('Lniru! Prn- /rsrur u/ hpiJnrn.dur;y: Jh.r.tur. ('..n- nrrricar Cancrr h.'piJrnunlur;y Unir. Yalc 1)oivcrvity Scbu.rl uf Medicine. New Ilavcn, (.'1. 1-1?KIU MI)RAD. M D., Pu.D.. Pru/rru.r u/ AlrJl, mr anJ Phtlrrlla{'alu~ v. S1an- furJ Un/versuy, and Chirl u/ MrJlrinr, 1'alo Alt.. V.A. Iluspiul, SunforJ, CA. JAY A. NAI)1'.1., M.D., Pru/rsu.r r./ A}rJlnnr. Plryslulugy anJ RaJluluAy, CarJiovas.ular Research Instilwc, Uni- rxuJECE' 'nT1E Review u( lung canur in ('unnccticul, 19)S-plcscm r ~ h C Mcahani.m uf mtnc uriJc aclivatlon u/ guanylalc cyclase 0 Kulc of cyclic GMP in sluuuth muvclc Ielaxauun Mcchanisms of airway bypcrirntahility vcrsity of Califurnia, San Francisco. Genctic basis for nicotinc respunse LX)NAI 1) l. NF.1 SON, PII U., Au{niurr Sptciruxupic .wJlcv of the ,rucracliun of Pr,.Jrl.ur u/ Chrrniury, ('Iark Univct- chulinclgic IlganJs wuh n/.urimc rcccp- sily, Wurccstcr, MA. tur pro/cins IIAKOLD 11. NI:WKAI.I. Au.hi- M.1). The rulc of prultaxv anJ anbprutcascs in The assuciatiun of inorganic dust depusi- , . ure PruJcuur u/ A1rJic8rr 'I he Juhns pulmunary culphyscma liun wuh pulmunary ncoplasu in to- , Huplins EJnivcrsity Schoul of Medicine baccu users . D'altinwrc. Eficcis of cigarette smoking on imnruno- I IoMdln proJucriun by human bronchial ymph{xytcs Assay of usygcnatcJ stcruls in buman b1uuJ vc»cls-a feasibility study Phagocytc mediated injury to tissues Studies of platclct and cnJuihc6al prosta- nuiJ pnKlucliun as p.».Iblc catJiuvas- cular t/sk indicators in s/nukcrs Inlcrachons bclwcat RNA tumur vuuscs and chemical carcinugcm Gcnctic anJ hiomarker studies of snwk- ing-aswclatcJ canccrs Chcnuslry and biology of cumplca callw- bydrates Interaction of Jrvalcnl cauuns with muJcl and brulogpcal mcmbranca 1cw FKANZ OI:SCH, Pn.D.. Pru/rssur u/ PharrnaruJugy; llraJ, Secrion un 8io- chrrrmicul PhurmocuJory, University of Main; Mairu, Wcst Gcrmany. BI:NDIC11'I' U. PAUI.I, D.V.M.. A.s.Ki- arr Pru/r.r..r u/ Pwh.duxy. Rush Pter byicri.n-St. Lukcs Medical Ccntcr, Chi- cago. UIsNNIS K Plil'I-KSIiN. Pr1.D.. Arrurunt Pru/ruur ,./ Pharmw.Ja}y, Elnivcrsily of ('ululaJu Schuul of Phanuacy. unulJcl. JUI IA M. IR)I AK, Dac'., M.D.. .Srna.r l.r— uurr in 1lh.nqruhulu}y. Royal Pus1- graJuatc McJ/cal School, Harnnrcrsmitb Iluspnal, London. WII 1.IAM A. I'KYQK. Prr 1)., Bu)•J Pr..• /.•.,w ../ ('hrnlurry, l.uuisiana Stalc Univcnuy, 1141011 Ruugc. 11.AK1 KANTASALO, M.D.. PrulllrlN NnJ CAAOmNn, Drl+.rrrnlrnr uf Public llrahh S.urnrr, Untvcnrty of Flclsinki, I/clsinki, FinlanJ. KONAI D F. KASMUSSIiN, Pu.D.. Ar- su{ iulr AJ)uncr Pralrssur in Curmrur- nuy anJ Fnnrunnrnral AleJu'inr- Uni- vcnily of California College of Mcdi- cinc, Irnnc. Metabolic fale :rnJ lus/culul;ical .ignili- cance of J.hyJr.Mli.ds JenveJ fruns pulycydic aruma6c AyJruca(bons uc- eorring in eigarette smuke Local «gulaliun of tumur invasion by husl-JcriveJ prwcmase rnhibuurs lnthalAes of gtINN)pc, K4 anJ chronic cigarcltc snwking .ul n{rut/nr anJ al- cu1W1 IrKlabulllnr in nlrCe InvcvligaUUn ..1 Ibc rnlr ..f rcgolalury' pcpti.ks tn M/man luag J/xasc Frct r,rJical :henusll) ul cip.rrctae snwke The Fmn/ah Iwin ('uh.al I-.dluw-up 1tuJy . 7 hc role uf cc11 .p.•cJlc tusinv In muusa hmg cA/cinugcncsu 101
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1 PRINCIPAL INVESTIGATOR OR INSTflUT1ON F:IIJiGN KIiM01.U-U'DONNF.1.1., Pu.D.. Prra.-r(,.d Krsrur.h A.\.NM'lutr. Ilarvar.I MrJrcal Schoul; bnr..n~nrur, Center fur llluud Kcscarch, Buston. JO/IN E. Klil•INIi, M.U.• Arsulunl I)i- r.•trur. wrhA-wariny Lurll: ln,lin,lr: Ai..K'iutr Pru/rsjar u/ MeJicinr, Uni- versily of ColorarJo Health Scienccs Center. Denver. III:KBERT Y. RIiYNOLDS, M.D.. Pru- /essr.r uf AfrJicinr Nrad, Palrnururry Srrtiun, Yalc Univenity Schuul of McJicinc. New llaven, CT. VIKGINIA 1.. KI('HMnNU. Pu.D.• Kr- scarrh Aasuciulr, 1'acihc Northwcst Kc- search Foundatwn, Scattlc. PIiTI•:R M. ROSS. Pr/.D.. Krt.enrrh A.w- r•lurr, l'he Kocke(clkr Univcrsity, New York. UNA S. RYAN. err.D.. Rrsrurcir Pru/cs- wr u/ MrJirinc• University of Miami School of Mcdicine, Miami, FL. B. V. KAMA SASTRY. D.Sc.. Ptt.D.. Pru/crsor ul Pharrrracofury, VanJcr- bilt University School of MedKlnc, Nashville, TN. ISHAIAHU SCHI: .CHTFR, PuU.. Srnn.r Lrtrwrrr in Bnn hrurirrry, The George S. Wrse Faculty fur l.ite Sciences, Tcl Aviv Univcrsity, Tcl Aviv. luacl• GERALD SI(KLAR, D.D.S.• Charlrs A. Brackcrr Pro/rssar ol Oral Palholury: //rad• Drparlrnenr o/ Oral Afrdreinr and Oral Pathology. Harvard School of Dental McdKlne, J3oslon. PKOJI<(:f' TITLE Purificatiun unJ funclional analy,rs of clastasc from juinca pis macroptrallcs Ba.ic mcchani.ms of IunA uqury Irum in- ha/cJ uaiJants Respiratory sccrcliuns in pulntonary car- cmoma: secretury component ul im- munuRlubulin-A as an eatly markcr of cpithclal dysfunction hfarAcrs of cpithclial cell Jysfunctiun in rcapiratury secretions of anukers lilastic tibcr micrulibrillar protein struc- lure DNA Jamayc and sclcctive maintenancc of animal kencs lnterac6uns u( Iwrmuncs willr cclls of the pulmonary vascular wall Influence of nicotine an tbc rclease of acuylcholtnc in the human placcnta and its implications on the fclal growth F.Ifcct of Ihiols and diwlhdcs on choles- tcrol metabolism Oral carcinu¢cnesis, vitaruin A and ret- inutds PR1NClPAL 1N V FS 'iK:ATOR UK INSITIUTIUN PKUJF:C C 7]'fLlr '17MU171Y A. SPRINGER. Pu.U., Ai- sisrunr Pru/rs.ur o/ Padwlury, llrt- SluJie% of macuuphaac .ubpupulatwns •"ol and Jilicrcntutiun using munuclonal 0 vard Medical Schuul, fioston. nntalllnlKs • 40 I{KI(' /•.lf'ANIIKIIXiP• I'u.D.. Atunudr r u/ All.rubwlury, Univpsny ul t'a/durnir 1, vine lrans(cr u( .pccdic mdtvtdual human ~ chruruuwmcs lu rccrpient cclb ~ • . DANIIiL SfbINIl1iK(i• A1.D., Pu.D.• Arlcrial JcrtaJaiun uf tuw Jcn.ny lrpu• ~ l9u/r..ur u/ AJrJhmr•: 1lruJ. Un•i..uun pruteins in vnv, ' - uf AfcluOuha Di,r.ur,'Ibe Umversity of ('alifurnia al San Dicgu, La Julla. ~ Chi OSSIiL M D macru- of Ihe lun m t l l t; . ~ rl, , . .. THOMAS P. ST Alydlcal Ont ulury Uni/, Massachusetts Gcncral Hospital. tloston. I). I ANSING TAYLOK. 14t.D.• A tuwiurr g y tmcuuna ana u phallc ('h.•mw.rsis uf macruph:.4cs _ _ P". ___ i,..J l'ru/n,ur u/ llwluKy, /IarvarJ Univet- su y. Cambr tJgc. M A. JAMGS TKAVIS, Pu.D.. tru/ruur u/ Biochernurry, The University of Geortia. Athens. EMIL K. UNANUE, M.D., AfalhncAruJt Pro/esrur u/ Irnmunoparhulury, Har- vard Mcdical School. Bouon. SII:PIII:N K VATNI:K• M.D., Asa/nrare PruJeuur u/ AlrJlcrnr• HarvatJ k1eJt- cal S.houl. Ncw f.nglanJ Regional primate Kcscarch Ccnlcr, Sanrlhbaxu. AIA.; .Ct...nrurr ur Alr.h:inc, 1'ctcr Ncnt Ilrixbaut Hospital. Bustun. I'1 II:K N. WAL11f, Pu.D.• Pru/ra..r u/ Al:Jnrn.•, Tcmplc University Schu.rl uf McJicirx, PhdaJelphia. GEORGE WFINUAUM• I'u D.. Nrusrr rnrist• Pulnn.nury f)ura.r .1r:ruen, AI- bcrt lanstein MeJical Center. Phda- Jclphta. 1. Itl KNAKI) WI:INJII'IN. AL1).. . Jr....,r .,f AJrdaurr uaJ l.n:n..arnrnlul ~.irmrt, ('.duutbia Unrvcraty, Ncw Y.n \. Protcolytic enzymes and inhibitors in em- physcma Physiupathulugy of normal and activatcd macrophascs Direct eBccts of nicottnc on twain circu- lation Intcraaiun uf plat.lch with cuaYulalwn frcturs IX anJ X Bruncbtualveolar lavaae .u( hunsan smuk- crs and rwnsmolcts: studies on cell cbemulaais, enzyme rclease and eellu- lar ultcastructura th•vclulwn.•nt ..( muo.0an..l anlJwJics to carcirrugcn-UNA aJJuas KOBFKT J. 1K1.AKF:W Pu.U.. Krrrunh Cytulmctics uf hctcrupluiJ suhpopulalruns A...r iatr Pru/rssr.r u/ Pwhulury, New by inratint SI(iM()ND A. WIif17h1AN, M.D. Ar StuJics ..( ph +guc) t.-iuJu: cJ nuuatwn York Univcrvity Kcscarch Scrvicc sru.uu Pru/c•.w•r u/ AlrJr:nrr, Ilcma- • Cnddwatcr Mensorial Huspilal Ruuse- ruloYy-Ontulu}y Unit. Mass.chuscRs , vch IslanJ. New York. GerKt,J Iluspndl, Bustun. Associare Pr.,- HANOCH SLOR Pu.D. The use of .p.•ciGe anuMwhe. tu munmtur AA1: WI-.NNAlA1 M• M.D.• .(...niut: Ntcutinc 1s iuh/buur ../ prua.t~lauJm (or- . , /rssuv u/ JJMINan Genrlics Sacllcr Ihe formauun .nd rentuval uf henru- Pn./r.u,r ../ (7uuru/ Pliy.i.Ja.cy wl roath•rt: L.:..Irtauun ul Ihc tnhtbu.wy , School of McJicine Tel Aviv Uni- (a)pyrcne aJJuits fnnn DNA of Jarru- K.u,.Irn.Lu ln.ruarr, HuJJin`c Huspir stcp anJ characlencatrwt u/ tlse earJw- , vusity, Tcl Aviv, laracl. atcd human cclls tal. IIuJJtngc. SwcJcn. vascul.r iruplicatwns DENNIS M. SMITH, PH.D., Aisiuanl Prulcssw u/ Birduriaul Surnrrs, Wcl- ksky Colkjc, Wclkslcy, MA. 102 Autunomic cunuul of pulmonAry surfac- tanl in Ihc aJult lung JOIIN 1. Wlt SON. 1'u U.. Pr,,. !. ....r ../ C. ll un.! Af.,l:. uL.r lt.,.l,.~ v. McJrcjl ('ullc}c of (:c.xtu. AurtWa. lh.• nad.rtian anJ esprc» ion u/ human n•I-:.utur~pan y:cnc wy/KUCr, thr.nr}h uwlciu/ar cluainr IU3
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PRIA1(a PAt. 1N V ESTIGATOR OR INSTITUTION ALV1N WINTERS. PttD.. Aarisranr Professor o/ Mrdicd Mkrob(oJogy, Vetcrans Adatinirtratios b/odica) Ccn- ta, Bay Pinca, FL. BRUCE A. WODA. M.D.. Associare Pro- /e.sor u/ Palbololy, Uaiversily of Mas- uctsurctu, Worccucr. STANLEY YACHNIN, M.D., Professor u/ Mrdicinr and Cbir/, Section o/ lJcm- arolosy/OncoJoly Tbe Univcrsity of Chicago Mediul &ater, Chicago. PROJt:(.'C Tt'n E ERecrs of smokina on the inhcrcnt inter- (cron levels in control and eanur pa- licnts: a pilot study Ccll surface munbranes, integral mem- branc proacins and cytoskclcton in lym- pbocytcs (rom: (1) young and old rau; (2) apoouncoua rat lympbomas Modcls (or tbe Patboicnesis of atbcro- sclerosis: A) biological elfects of osy acnatcd stcrol compuunds; B) mcvalonmc acid rutd cbolcucrot biosyntbcsis and the bioaynlbcais and regulation o( ccll growth Completed Projects Following is a list ol the priacipal inveatigators<, or inatitutioni, of projects that have been completed prtor to the pcriod coveted in this Report. Seberal of the individuala named are doceased.'Ihe titlea and a05Jia- tions listed arc thosc in effect at tbe time the work was completed. LEO O. ABOOU. Pe.D.. Pro/essor o/ Riochcmialry and Brain Rrscarch. Ccn- tet (or Brain Researcb, The University of Rocbcstcr Mcdical Center. Roches- tcr, NY. MARIO D. ACkTO, Pn.D.. Associar. Professor o/ Pharmacolafy, Medical Col/c e of Virainla. Viraicua Commoo- wcaltt Uaivctrtty, Rktsawod. CLARENCE M. AGRL'SS, M.t)., Auo- ciarr Clinical Pro/etsor o/ Alydicinr. Univcrsity of Califoraia Medkal Cen- Icr, Los Angcles. ANT)tONY A. ALBANESE. Pw.D., Di- rfclor O/ LAboralw/rs, Tbe Burke Re- habilitation Center. White Plains, NY. ANTHONY P. AMAROSE, Pn.D.. Jn- l/7Ycl,M in Obarerrics and Gynecology. Tbe Albany Medical Colksc o( Union Univcrsity, Albany. NY. C. T. ANOt:LAKOS, M.D.. Pn.D., Pro- /essor o/ Physiology, Bostoa Uaiversity School of Mcdicine, Boston. 0. MURRAY ANGEVINE, M.D.. Uni- vcrsity of Wisconsin School of Medi- cinc, Madison. JOSEPH C. ARCOS, DSc., Professor o/ Mcdicinr, Tulane Univetsity Scbool o( Medicine. Ncw Orleans. ALAN K. ARMITAGE, Pn.D., RruarcA Dirrrror, Iladcton Laboratorics Eu- rupe, (lanoaate, North Yorkshire. P.ns- land. MARILYN S. ARNUTT (RASCO), Ih/.U., .(asnrim! Biulo,rur and As.:sronr Pru/euw u/ Bi.dogy, 7be Univeruty of Tcs.a Sysrao ('anccr Center. M. D. An- dcrsun tluspita and Tumor lnuitutc, I luustun. STEPHEN M. AYRES. M.D.. Diriclor, CardioPulaionary Loborarory, SaiM Vioccnl'a Huspital, New York. OSCAR 1. BALCIIUM, Prt.D., llaarlnss Pro/rssor o/ Mrdicdne. Uaiveaity of Souwhcrn Cali(oraia School of Mcdi- cinc. Los Anacks. FRGUIiRIK B. BANO, M.D., Professor and C/wir.nan. Drparr.nent u/ PalAo- blolugy, The Johns ttoptins Univcrsity School of Hysienc and Public Health. Baltimore. A. CLIFFORD BARGER, M.D.. Robert Henry P/tiffrr Professor o/ PAysiol- oty, Harvard Medical Scbool, Swro& BRODA A. BARNES. M.D.. PN.D, Pro- /essur (Adiliare) o/ PJtysloloty. Colo- rado State University. Foci Coltis. FREDERICK W. BARNES. la., M.D.. Asswiaa Professor o/ Medicine. The Jobns Hopkins University Sciool of Medicine. Baltirnorc. T. C. BARNES. D.Sc.. RrsrarcA Scirn- rl.r, P)siiadelp0ia State Hospital, PLila- deJpbia. CARL O. BECKER. M.D., Associae Professor o/ Pathology. Cornell Uai- versity Medical College. New Yort. R. FREDERICK BECKER. PN.D., Auo- ciase Professor ol Anatomy and Direc- rur. Laboratory o/ Prrinara) Science. DuLc University Medical Ccnter, Dur- Aun, NC. RALPH S. BECKER, PN.D.. Professor ol Chcmiury, University of Houuon, Houuon. BENJAMIN BEI.i., M.U., Dufcl.w firurr- ilw, Nwmalnv Ariny SlwJy, Vctcr- aas Adarinistration Outpuicnt Clinic. Bouun. DOMINGO 161. AVIADO. M.D.. Pra/ra- nor o/ Phrnnaculury. University of Pennaylvanra School of Medicine. Pb.l- adelpbia. SAMUEL BELLET. M.D.. D/rrcror, Di- ris,un o/ Cardiuloey. Philadelphia General Hospital, Philadelphia. 104 105
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c BAKUJ BENACERRAF, AI.D., Fahyan Prufessur and Clwirnww. Drpurtnuru ul Paholuly, Harvard 1lcdical Scbool. Bostoa. WILLIAM F. BENEDICT. M.D.. As+/s- lanr Pru/rssur uJ Prd/arrlca. Unfvcrsity of Soulhern California School of Mcdi• cinc, Division of l/cnsatulogy and Mcd- lal (7coerics, CLildrsa's 1(ospllal of Los Anscks, Los AnScks- JOHN A. BEVAN. M.D., Pru/tssur a/ Pharn,aa•oloty, University ol Cali(or- nia School of Medicirx, Los Anselcs. BUDIiDEV B)IAGAT, Pu.D„ Pru/tssur u/ Physiulury, Saint Louis Univcrsily Schuol of MCdtc1M, St. Louis. CESARE BIANCIFIORI, M.D.. Divisi.rn ul Cwctr Resrara•h, University of Pcrusia, Pcrugia, Italy. HYLAN A. BICKEKMAN, M.D.. dssist- an/ Piu/rrsur o/ Aledicine, and AL. VAN L. BAKACH, M.D.. CunsnUanr in Aledicine, Columbia University Col- lege of Physicians i Sur`eons; Gold- watcr Mcmorial Hospital, New York. RAYMOND BOSSE. Prn.D., .luuciule D.rtclw, Nunnutive ./rinr Sn.Jy, Vct- enns Administration Outpalicnt Clinic, Boston. TOM O. BOWERY. Pu.D., Rtstwch Pro/tsror. Prsrlcide Rtsldwt Labora- rory. North Carolina State CoUcjc, Raklah, GEOFFREY L. BRINKMAN, M.D., As- sociatt Pru/nsor o/ Medicint, Wayoe Sutc University School of Medicine, Dcsroit. ROBERT E. BKOOKS, Prr.D., Associatt Pru/tuur of Paholury. Univcrsily of OrcjSun Mcdical School. Portland. BARBARA B. BROWN. Pn.D., Chief, lisptnw.tntal Psychiatry, Vacrans Ad• miniatralion Huspital, Sepulveda, CA. WIl.1.lAM 11. CARNES. M.D.. Univcr- JULIUS Il. CUMKOE, la., M.D.. Direc- stly of Utah CoUcje of btfedicine. Salt lur, CurJiurrucrlar• Hruarch Institwr, Lake City. Univcnity of California Medical Cea- tAKCUS N CAKKOLL la PoD ter, Sae Fr.nciscu. h 00 ~ '~ ChirJ, Diris/un of PAarrnacololl. TDa '. DEAN 111. CONNORS. M.D.. .1 rsucia! Qir I/rookdak Huspital Centsr, Drooklyn, DYaefur, Drp.r brrtnr u/ Laborarary ~ NY. M.lkinr. St h(ary's Husqt.l, Madiwa, WI. t ) W11.L1AM ALVIN CARTER, M.D.. Assisranr Pru/rs.w oJ Medicine and PIIILIP COOPER. M.D.. C/inical Pio- ~ Alicrubiulury, The Johns Hopkins Ual• lessor ul Surrery and Dirtcrw, Surgi. vcrsity Schoul of Mcdicine, Baltimore. •nl Lohoraa,ry o/ CclGdw Physuololy, LIsOPOI.D K. CFKECIiDO, Pu.D., Pro- /nwr of llioihe,ni,lry and Nutritiun. llnivasily u/ Pucrlu Rico School o( Atcdicinc, San Juan. JACK CIIALON, M.D., Auuuwr Pru/r.• s.,r uJ anr.rhu.uluYy. Ncw Yutk Uni- venity Medical Centcr, New York. Albert Ernslun Collcle or Med.ane of Yesbiva University; Ch,ct Surj.cal Strnrt, Vctcrans AdmniNraUUn ilos- pnal,'1 Ac Utuns, NY. PAUI. T. COSTA. Jr. Pu.D., Assocwrr Pru/r,sur u/ Psy.holuly. University o( Ma»acbuxtts at Bostun, Durchester. JUHN F. CRAIGHEAD. M.D.. Pro/rs- Pro/tr- RAYMOND K Pii D BROWN sur u/ Pathology Umvcrsity uf Vcr- . . .. . sur u/ Clinical CJncolory University of CIIII.DRI:N-S HOSPITAL OF LOS AN- . nlunt CullcSc of Mcdicmc Burlington. , Wiscunsin Medical School. Madison. GELES, Los Angeles. , Aarisranr CRAIN D. ROBERT L PH JOSEF BKO7.EK, PN.D., Pro/tssur and SANI-UKU CIIUI)OSIL M.D., A„islanl , . . . rru/r,.ur u/ Sw-iulury Universily of Chairman. Dtparrnrent o/ Psycholosy, Pru/rasw uJ MrJicine, Tufts Univer- . Chlcatu Chicago. Lehigh Univcrsily, Bclhlchem, PA. sity School of Mcdicinc, Buslun. . ' IRVING P. CKAWI UKD ALD. Prolrr BIO•KESEARCH CONSULTANTS Assislanr SUE BUCKINGHAM M.D. IIOPRA, PruJrs- NAIfPK A1. C . . sur a.J Chuinnnn Drp,rlrnrat o/ Ali- . INC Cambrid&e MA. , . PruJessor of Ptdiarria Columbia Uni- sor .,/ (7amuuy, Nu.th Cawlina Ag• . crobiololy Univcnity of Iowa College ., , , versity CoUe`e of Physieians A Sur- ri.ullural and Tcchnical Sl:de Univer- . uf Medierne Iowa City BIO•KESEAKCH INSTITUTE. INC.. &cuns, New York. sity, Grecmburu. , . CambriJSe MA f. 77AIUfHI' CRCXKF.K. A1.D., Pro/rs• , . A. SONIA BUIST. M.D., Associare Pro- WI1.1 IAM G. CLAKK, Pu.U., Dirtcrur, .,..r u/ Ale.hrtnt. Univcrsily uf Cali• Assoclate Can- FRED O. BOCK Pu.D. /essor o/ Medicine and Plrysiolury. rayr'H.,phurn.uaulury 1ltKUrrh furnia Cullcye of A1cJocmc, Irvine. , , Biolorical Sro- « r Restarch S.KMlu Univcrsity of Oregon Hcaltb Sciences rury, Veterans Admini.lratiun Iluspilal, , riun, Roswell Park Memorial lauilute, Ccatcr, PurAand. Scpulvcda, CA. CARRUI.I. f. CKU)S, M.D.. .I.aanur Springvilk NY rru/t....r u/ AIr.G.u,r unJ lJn,nun , . BENJAMIN BURROWS. M.D., Assu- IiANS -T. C:LAKKIi, D.Sc.. Pn./r.,ur u/ Plq•.i..lu4 r: Dnr. t„r, 1n n„n ../ GUENTHER BODEN. M.D., Associutt ciute Professor of Mtdicine. University lli.a'heu.ulry, ('uluurlria Uuivcnity nlulNlry AlrJau.r. C)nrvcrwl)' u( (*ali• Proftssor of Mrdicine; AssiNanr Dirra•- of Chicago. Chicago. Collcte of Ph)'sician. S SurQcun., Nrw furnia StLuus uf McJ,.m., Dav.s. Gtntral Clinical RrxarcA Ctnrei ror York. , Tem UalvsraUy Hsaltb SwMoces Cea- E. M. BUTT. M.D CA/tJ ParhoJo /st, ~ CECIL E. CROSS, Restach Dtpartnrtnt, tal. Los Angsla Coumy General Hosp JAY D COFFMAN D S M ( e ilal Burb nk ('A ts~ Ji Jo tar, p4fa.:• r Los Anyela. . -: oat ; . . rd JIu.J, PaipAtra/ Yaxvdar Dqurrerrdrr~ p , , u a . r ~~ d HERMAN V. BOHN/O, Pw,D. dtaq Pro- Pst D RICHARD U BYERRUM Univcrsily Iluspital, Uuuun. t ~CRUAIPACKFK, Pu.D,tru nd IlocAtnr- r f Cii rnl a D . ., . . Irww anJ CAannwn, Drpwrnanr uJ tpar/nmtn o t y a r /tuw ol Chrnr/sary. Michiaan Stata : GnrYonnrtn/al ulutiun and Or Pu a• 1ury, Spiodlctop ReasarcL Ceaur, Lea• Universit East Lansin A111 N 1/. CU1I1:N, A1.1).. I'u.l).. A.- . p l inglow, KY. y, s. ..,.iart Pru/tuur u/ Alr.brinr. C/dtf, nisndc Biuluq. Unrvcrsity of Culoradu, SISTER M. IiMILY CAHILL. Pu.U., ruln.unory Srct.on. Temple Univeraty Uuul.kr. JAMES F. BONNER. Pw.D., Profesror Chairman. Departn,tn! of Chunutry. Scicnces Ccntcr, Philadelphia. 1A1U ALUNKI' U.iA1ON 19/1) 1rr- ol BioArty, Califor.ia /auiWte o/ Rcsis Cullcgc, Wcslun, A/A. , , . lwrr un Iruhrupululy; Rramah Asuu- Tccbwiogy, Pasadcna. BRUCE F. CAMI:RON, M.D.. Prt.D., UANIh1 CUI11'N, D.V.M., A.- u,ranr Prolessur .,/ rrlrri,uuy Fpi- untr .n Altdic'a! Inlbrup..l.•ry, Pea- WALTER M. BOOKER. Psr.D. ProJrs- Howard IluWs Instilutc, Univcnily Jr,nwlury unJ Publ.c lleulr6 Univer- budy Museum, HarvarJ Urdveraly, ' sor and UtaJ. Drpar/mtnr o/ PAarnm- uf Miami School of Mcdiciac, Miami, , sily of Pennsylvania Sch.wl uf Vclcr- ( a,ulwidt;c. MA. colusy. Howard Uaiversity, Waahins- FL. mary Mcdtcinc, 1•hil.dclphia. 711UAIA!s K UAWUf:R Asucwrr M D loa. DC. . , ., P AI J B U FKAN<O1S M. BOOYSE. Pn.D.. Stniur lnrtuilatw, Micb.el Reese Research Fouadatioa, Chicaao. ELKOY T. CANTRELL, Pu.D., Chau- nran, Drparln.tnr o/ PAae,aculury. Teaas CoUeje uf Ostcopathic Mcdicinc, North Tcaaa State Univcrsity, Dcnton. ALLAN C. CULLINS, Pn.D., A.,wiare Pru/tssur u/ Phwrnacolury. lnsrJWtc fut Bchaviural Genetics. Univcrsity ul Colorado, Buuldcr. ru/nsar u/ t a.ne, ustun n.ver- sily S.huul uf MsJicine, Buuun. K. F. U.\WSUN, PH.D., Pru/t.,..r u/ Hur- uny, Coluu.bia UnivuNty, New York. 107
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I JOHN P. DELANEY, M.D.. Ptt.D.. As- socwrc Prafeuur of Surgery, Univcrsity of Miancsota, Miancapolis. ANDREW S. DIBNER, Prr.D., Eirc- urive. Psycho-Rcstarch, The Age Ccn- tcr of Ncw England. Inc, Bouoa. EDWARD F. DOMINO. M.D.. Pru/ts• sor uf Pbarntacufugy. University of Michigan. Ann AMbor. RALPH L. DORFMAN, Prr.D., Director u/ Lobwarorits, Worcester Foundation for Eapcrirncntal Biolop, Shrcwsbury, MA. H. FKIiU UOWNEY Pn.D., Asrutu.nt Pru/tssur uJ Physiuluy. Univcrsity of Tcaas Ilcahh Science Center at Dall.u; Direclor, Cwdiuvasculw Rc,eurrh, Car• diupulmunary Inslitute, Mcthodiu Hus• pital uf Uallas, Dallas. JAMES J. DYAR, Prr.D., Assisraiu Pro- leasw u/ Biololy, BcUarmine CuUcjc, LouisvilJc, KY. RICHARD Jl. EARLE. M.D.. Chie/, Prdntonary Function Laboratwy; As- sulanr Pru/essor o/ Mcdicint. Univcr- sity ol Chicago. Cbicatu. JOHN W. ECKSTEIN M.D.. Asaisrant Prufessur o/ Jnrtrna} Medicine, Statc University of lowa College of Mcdi- citse, lowa City. BERTRAM EICIIEL. D.D.S.. Director. fnstirurc o/ StonraruJosical Research. Scicacc Resources FarrrJatiun, Water- town. MA. HYMAN ENGELBERG, M.D.. Auend- ing Physician, Cedars of Lebanon Hos• pital, Los Anyelcs. CAKLTON K. FKICKSUN. Pu D.. Pr„- /cs.n• u/ Phur.nu.aduay. The Univcrsity uf 'hetas Cullcsc ul Pharmacy. Austin. V. GFNIi I kWIN. Iht.U.. Pru/r~wr ol Pb.urnur.du+y; Dtun• Univcnity af CrrlutaJu School of Pbarmxy. BoulJcr. IiENKY J. ESUI:K, Prr.D., Rtstwch !nt- ruurruJuY,rr, Masoa RcscarcL Institutc, Warcester, MA. WALTER B. ESSMAN. M.D.. Prr.D., Pru/usw a/ Pnycholury and B,ochtn,- iarry, Queens College of the City Uni• vcrsity of New Yort, Flusbing. JUIIN R. IS'ff;R1.Y, M.D.. Assuciate Pru/taur ul Pdtholugy. University of Chicago Pritzker School of Medicine. Chicago. HUGH E. EVANS. M.D., Doecrur, Ur- parl,ntnl u/ PrJn,tna's, Jewish Iluspi- tal and Mcdical Centcr of Broutlyn, Broul,lyn, NY. HANS J. El'Sf NC-K, Pu.D.. D.Sc.. Pru- /tssw u/ P4yrhulu.y, Instirute of Psy chiatry, University of LunJon, Lon- dors. HANS 1.. FALK, Pu.D., AJ/nucr Associ• art l'ro/rstur o/ Parhuluty. University uf Suullrern Califurnia ticbuol of Mcdicinc, Lus Angeles. DANA 1.. FARNSWORTH. M.D., lltnry J:. (IlnYr P,u/t)tur u/ llyLitnt and lhrt,lur, U,uversuy JJrold, Strv,cts, 1l:rrvarJ Univct sny, C nnrbnJYc, MA. GAD FEINSTIi1N, Pu.D.. Senior Lec- natr irn Biw-htrni,rry, Thc Geurgc S. Wise Center of Life Scicnccs, Tcl Aviv Univcrsity, Tel Aviv, Isracl. FRANK C. FERGUSON. la., M.D.. Chairnwn, Dtparunenr u/ Pharma..d- u`y, Tbc Albany Mcdical College of Union Univcrsity, Albany. NY. T/ILODOKE N. FINLEY. M.D.. Direc- tor. PuLuunury Ruearch Laburatury, Mount Zion Hospital. Sao Francisco. WILLIAM I. FISHBEIN, M.D.. Chie/ of E~idcuuoloQy, Chicago Board of Hedtb, Chicago. EDWIN K. FISHER, M.D.. Director u/ LnGuratwin. Sbadyside Huspital; Pro- /tssur u/ Pathology. Univcrsity of Piusburbh School of Mcdicine, Pitu- burgb. RUSSIa.I. S. FISIII:K. 1.f.D., University of MarylanJ School of Medicine. Bal- ti,uurc. WILLIAM 11, FIS11h1AN, Pu.D, Prru- dtnt, La Julia Cancer Rcscarcb FuunJa- liun, La Ju1a, CA. B. 1.. FRF:F.01 ANDEK. M.D.. Durctor ul ('anlrr Rrstwch, Mount Zion llos- prtal and Medical Center. San Fran• ciscu. FREULKIC A. FKENCI/, A.U.. Dnec. tor u/ Cw,ctr Chernurherupy Rt,twch. Muunt 'LGUn Ilosprlal and Mcdical Ccnacr, San 1 ranciscu. 108 JACK FREUND. M.D., Ar,uranr Pro- ltssur o/ Pharmacology. McJical Col- Ietc of Virginia. Richmond. LAKS FRIUL'KO, M.D., Pru/essur and Chnirman. Department of Environ- nmenral 1lyRirnt, The Kar'olinuka Insli• lutc, Stockholm. GII.UI?KT 11. FRIUDI:LL, M.D.. Chief of Parholua•y. St. Vincent Hospital. Worustcr, MA. (iAKY 1). 1•KIEDMAN, M.D., As.i.ruN Dirtctur, Drpwtmtnt of Altdical Merh- ods Rrsrarch, Kaiser Foundation Re- sca,clr Insrilulc, l)a11.nJ, C A. I1. IIUUII /•11D1?N111?KU, M.D.. Pru/u- sor a/ M.Jnnnr, Univenity of Cali- lutnia Mcdical Cemcr, San Francisco; Pr,r/rs,ur u/ Bactrnoloty and Imntu- nulury, Uuivcrsily of Cahfurnia, Bcrtc- Icy. AKIIIUR FURST. Pu.D., Direcrur, !n- sriturt o/ Chemical Biolu`y, Univenity of San Francisco. San Francisco. MURRAY U. GARDNER. M.D.. Asso- r•iart Pru/tssor o/ Pathulugy. Univcr- sity of Southern California School of McJicinc, Los AnSclcs. GEORGE O. GEY, M.D.. Direcrw• Fin- nry-NouvU Can, er Research J.ubora- tury; Assocunt Pru/nsur of SurRtry. Thc Johns Huptins University School of b(cdicinc, Bahimorc. RONALD W. GILLETTE, Prr.D.. Dirtc- tor, Basic Science Rnearrh Unit, Can- cer Ccnlcr of Hawaii• University of Hawaii al Manoa, Honolulu. GI?KAI.D 1. GLF.ICH, M.D., Cu.anltunr in Afrd,cant, Kescarcb l.alwratury for Allersic Discases, Mayu Clinic anJ FounJation; Prufc.uur .,/ lrurrnul AfrJic,nt and lnu,u,nulucv, Mayu MrJ,cal J.huul, K.whcstcr, MN. '1'110AIAS M G(K'Kli• M 1), Asrwiarr Pr../ru.u uf Prnvnurt Altdir,nt und Cwnn,urtmty J/rolrl,, Se/un Hall Cul• IeRc of A1cJ,cine and Ucnustry, Jcrscy City, NJ. 1)AVII) AI. (iOl DI;NUI:KG• Sr.D, A1.D.. .1„u„urt Pr../ruur uf Pwhuf- ujy. Tcmplc Un,veraty Ilcrlth Sci- cnccs ('entcr, PhJaJclpbia. PAUI (;r)I I)IIAUI;K, D.OS.. A...riutt Pr,./: „ur ,./ I IarvarJ S.h.wl of 1).•nta Mcrhunc, Uuston. 109 r-1 I.EUNIDE GUI DSII'IN, D.SC.• Asso- ctutt Pr,./rarur of Psych,atry, Institute lur Mental Hcaltb Scicnccs, Cullcse of Q Mcdicinc A Dcmi.try of New Jerscy, ~, Rutgers Medical Scbool, Piuauway. ` IRA GOKF, M D, Pro/t,sur uf Pwhol- oKy, Boston University School of Mcdi- ane; Chie/ u/ Lahuratury Servict- Vetcrans Administration Hospital. West kuabury, MA. JOHN W. GORKOD. D.C.C.. Ltcrurtr in lAup/urmacy. Chelxa Colletc, Umver- sity of London. London. OF.K7 kUU1? Y. GU7'ISCIIAI.1., Prr.D., Ar,isranr Pru/trrw u/ Bn.rht.nirrry. Culumbra Univrrsity Cullcte of Physi- cians i Surficuns, New Yort. A. ('LARK GKIFFIN, Pu D., Htad, Depuruncnr of Biuchrnusrry. M. D. Anderson Hospital and Tumor Insli- tute, University of Tesas Medical Center. Houston. ARTIIUR 1.. GKOSS, M.S., Teniw Bio- che,nirt, Southwest Research lauitutc, San Antunio, TX• MORTON 1. GROSSMAN, M.D.. Prr.D., Associate Clinical Pro/trsor o/ Mrdi- rine. University of California Medical Ccntcr, Los Angeles. CARI- C. GRUIIZIT, Af.D.. Pu.D., As- suci,ue in Physiolo.y and PharrnacoL oAy. Univcrsity of Pcnnsylvania Grad- uale School of MeJicine• Philadelphia. JOSEPH 1. GUARNERI. Prr D. Atrend- ing Mirrob,ulax,sr D,recrw. Microbi- ology Laborarories, Long Island Jcw- ish-Hillside Medical Centcr, Qucens Huspiul Center Affiliation, lamaica, N Y. ItODA A. GUIKGI.I-. PN.D, Ar,.n-iutr Pr.•/e14N ../ ('.,muu.o,ry unJ J:'m•oon- .ntnral AItJn tnt, University o( Cali• fwuia Cu11cRe of AteJi.ine, livinc. FKANIC F. GIff)IKIF, PnD, Pr../tr- rw u/ t'nturnulux y. North Carobna State College. Kaleish. 11. U J/AAG, AI D. Pn./tuur u/ Phur. n,•n.dury. McJ,cal Collesc of Virginia. KiclunursJ. F. J. HADDY, M D., PuD, PrnJtssw unJ Chnuman. Drparunrnt u/ Pbyu- .J,..•y. Uruvcrsuy of OU.Mnna McJical l.'cutcr, Ullahunta l'ny.
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I I JOSEPH H. HAFKENSCHIEL, M.D.. Dirtcrur, CarrGopulsnonarY f/nir- Tbc Lankcnau Hospital- Associa/r in Aledi- clnt. University of Pcnnsylvania School of Medicine. Philadclphia. MARGIT HAMOSH, PK.D. Rtsrurch Assuciarc, Department o/ Physiulaly and Niuphysfcs, Gcorfelown Univcr- sity Schools of Medictae and Deatis- try, Washington, D.C. BERNARD HANES, PHD., Pro/asor of Health Science. Cafiforrsia State Univcr- aty, Northridga RICHARD 1. IIAVEL, M.D.. Asrisra.u Prolesxrr u/ AleJ.cinr, Unrveruty of California Medical Centcr, San Fran- cisco. IIERBERT R. 11AWI71OKNC•, M.D.. CJwrrrnan, Department u/ Surgery. University of Pennsylvania Graduatc School of Medicine. Philadclphia. JOHN A. HAYLS, M.D.. Associare Pathologist. Mallory Institute of Pa- thology, Boston City Hospital, Boston. CLARK W. HEATH. M.D., Pru/t»or o/ Medicine and Dirtcror o/ Health Strv- icts, Tufts Universily, Medford, MA. PAULINE HEIZER. Pw.D., Rcstarch Assuciau in CyroloFy and Cyruchc.nis- rry, San Franc+sco lastitutc of Medical Sciences. San Francisco. CAROL J. HENRY. Prr.D., Director, De- prrmtnr o/ EAptrlrrrtntal Oncology. Miaohiological A.aociuu, Ioc, Be- Ihesda, MD. HERBERT B. HL:KSCOWITZ, Pn.D.- Assuciart Pru/rraw uf Micrubiulugy- Gcurgetown Univershy Schools of Mcd- icinc and Dcruis(ry. Washington, D.C. LAWRENCE L. HESTER. Ja., M.D.. Professor and Chairman, Dtparrmtnr o/ Obsrerricr and Gynrcolurr Medical College of South Carolina. Charkuon. EBBE CUKTIS 110FF, M.D., Prr.U.. Professor and Chabman, Division of Psychiatric Rtsearch, Medical Colkge of Vrrglnra, RKhmond. RUSSELL L. HOLMAN, M.D., L.ouisi- a.u Sta(c University School of Mcdi- cine, New Ork.na. OLE A. HOLTEKMANN, M.D., Rt- uarch Sticnriu, Lobund Laborarury, Univerut~ of Notre Darnc,_ Nwrc Damc,1N. •RI:UDY tJUk/UUKG1-K. k1.D.. Prtir- drnr unJ Duc.v..r, Biu-Kcscarch lnsp- tutc, Irk., Caml+ridgc, MA. KUHIiK I' W. HULL. Pu.U.. Pru/asor o/ Uiulugrcul Scitnccs, Hurida State ' TfA1.1.A11 KAPPAS. M D., Pro/ruur and Senior Physiaran, The Rocke(cllcr University. New York. IIKATCII KASPAKIAN, M.D., Ai.rist- anr Dire.l,n, CrrJa.raiculur LaAura- Hrhnc- rury; Irurnn rur in Afedicinr HAKLES W. I..BELLI?, Prr D., A„i,r- anr Piu/rrior ul Envwun.nrnrul lly- Iricnt, Jclfc(son Medical College. Phila- dclphia. AARON l. LADMAN- Pn.D.- Pro/trsur and Chairnran, Department o/ Anar- C C C T I ~ ""Ir Nzr e-! L N ti..f ~ I alabha»cc. University. , munn Mcd.cal Collcgc and Hospital, umy- The University of Ncw Mcaico ~ RONALD K. HUTCHINSON, Pr/.D.. Re- Philadclphr... School of Medicine. Albuquerque. search Direcrur, Foundation (ur Bcbav- MI Ii1.111U KA7Z, IhrD., Arsu.-iure Pru/rs- THOMAS C. LAIPPLY, M.D. Pro/u- ~ .t:.. . iural Research. Augustu, ICI' RESEARCH INSTTRIfE, Cbicago. iur u/ Sucinlury. University of Chi- cagu, Chicago. wr u/ Parhology, Northwestern Uni- versuy Medical School, Chicago. .r:,.. ALP/ IONSE l. INGENITO. Ihr.U., Asru- ciarr Pru/ea.ur u/ PhurrnnculuAY, lia.t Carulina tlnivcrsitY Schoul of Mtdr ctne, Grcenvillc, NC. IIAKKY l.. IUAI.'lllld, AI I).. AutnJuir; ParhuluciN, Lcnos Ilill Iluspilal: Chnr rul Pru/tssur u/ 1'urhuluAy. Culuurbir Univcrsity College of Physicians i Surgcuns, New York. (jF:OK(iL' JACOBSON. M.D., Pruftrrur unJ HeuJ- Department u/ RoJruloly. Univcrsity uf Suutbcrn California Schuol o( htcdicinc, Los Angeles. JERKY HART JACOBSON, M.D.. Di- rtclur, Dn•rsion of Elecrruphysiolury. New York Eye and Ear Inhrmaty, New York. JULIUS Il. JACOBSON 11, hl.D.. A»a- erart Pru/rsui ul SurYery and Drrecror of Surgical Research, University of Vermont College of Medicine. Bur- lingtoa. MURRAY E. JARVIK. Pu.D., .lssuciart Prufusur of Pharmaculogy, Allxrt Ein- slein College of Mcdicine of Yeshiva Univcrsity, The Brona, NY. DAVID A. IOIINSON, Pn.D.. Auurunr Prufessur of Biuchenrisrry, East Tennes- see Statc University College of Mcd- icinc, Johnson City. OSWALD R. JONES. M.D., St. Luke's Iluspital, Ncw York. WILI.IAM 1. 1t1SKO, 10t.U., As.ui.urr Pru/tssur uf Phurrnucnun-4; lhrtrvur, Clinical PhurnwcuMnrrtcr l.aAururory, Millard Friluww Iluspttal, BuUalo. ANDKEW A. KANDU7SC1f, Pn.U., SraQ Scirnrirr, The Jackson Labora- lory, Bar Ilarbor, ML:. ARNOLD R. KAPL.AN, Prr.D.. Duec- ror, Laburwury of Medical Gcncr.cs. Cleveland Psychiatric Institute and Hospital. Cleveland. SIfIK1.L:Y L. KAUFFMAN, i,I.D., Pro- /essur u/ Parbulugy, Slate LJniversily uf New Yuik Uuwtulalc Mcdreal Ccn- tcr, Bruuklyn. ANl'Ll. Kli)'S, 1'u.D.- Dirtcrur, LuAuru- rury u/ Physiululical Jly6ienr, Unrvcr- suy u( Minncsuta School of Public licalth, Minneapolis. JWEI'H H. KIKSNER, M.D., Professor / A/rJ.cine, University of Chicagu School of kledicinc, Chicago. EDWARD 1. KLAIB[K, M.D. St+uur Scienrisr, Tbe Wurcestcr Fuundatiun fur Eapcrimental BiolugY, Inc., Shrcws- bury, MA. 11'.KOk11: K1.1'INIi)(MAN- A1 U.. Pru- /rir..r und l'burrm,u., !)rp..rrnaiu .,/ P.uhalugy, Muunt Sinai Sctwul of Med- iune- New Yurk. PI•.TEK li. KNAPP, At.D.- Research Pru/es...r of Pqchiarry, Buslon Uni- vcrsny School of Mcdicinc, Boston. KI:NNI• 11/ P. KNUDTSUN, M.D., Uni- versity of Washington School of Medi- cinc, Scrtllc. ALVIN 1. KOSAK. Pn.D.- .luuciart Pru/trsur u/ Chnnurry, Ntw York L/nivcrsrty, New Yurk. K(>I11:KT A. KUHN. M.1), Arrur.urt 1•ru/ruw. l)tvr.mn a/ Nturu.urr;try, Ncw lcrscy State College uf Mcdictne, Jcsscy City. SIIG KUI.I.ANI)FK, M.D., Pru/r„ur anJ Clmornun, 1)rpurr.nrnr ../ OArrer- r.cs and Gynecoloty, University of Lund. l.uod- Sweden. MARVIN KUSCIINI:.K, M.D.. New York Unrvcrsity Medical Center. New York. MICFIAta. li. LAMAI- kt 1).. Pru/tuor uf Purl..d..ly, New Yurk Univcrsny Mtdi- cal Ccntcr, New York. 1'AUI. S. LAKSUN- Prr.D- NuuR Pru/e.- sur aJ Pluomuculury, klcdreal College of Virginia. Richnrund. KOGI:K K. LARSON. M.D.. Chief of MtJicine. Fresno County Hospital. Fresno, CA. GU5fAVE A. LAUKfNl.l, M.D., Chit/ ,ccstcrr,rMA,St. Vincent Hospital. Wur. JOSEPH A1. I ALIWI:RYNS, M.D.. PK.U., Pru/rs.ur UrJurunas and Charr- nran. Department u/ Padroloty and Microscupic Anarwny, Esperimcntal Laboratory of Putmunary Hnlopalhol- ogy, Katho6cke Universiteit tc Leuvcn, Leuven, Belgium. JAk1ES C. 1 PF, Pn.D.- .4isi.runr Pro/ts- sur ../ lli.+ hemi.rry. Saint l.uuis Sihuol of M.Jicmc, St. I.urus. EDWARD I I:E'fE, PtrU., D.Sc., Pru/rr- wr .•f Chrnmsrry, University of Minne- aota. Atinncapohs. RICHARD A. LERNER. M.D.- .lrsucart Mtn.btr- Scripps Clinic and Rcscarch Foundation, La Jolla, CA. CECILE L.liUC1iTENBL:RGF.R, Prr.D., l1tuJ, Department u/ Cyr.n hnrurrry, Swiss Inwtutt for Eapenmenral Can- ccr Kcscarcb, Lausannc, Swiucrland. JAY A. 1.1[VY, k1.U., Ass.nune Pr.r- /trrlN u/ AfeJ.. mr1 Rr,run h As.,.cb are. Carr•e( Rcseareh Inatirute, Unr- venhy of California School of Medi- cinc, San Francisco. P11fJl D. 11'WIS, AL1)., Jrn.,u Lnrrurtr rn lluraµu)ud.r.~r, Ku)dl Pustgraduate ktcd.cal S:huul, Harumcratuth I/ospi- 1/1, 1 ond.m. III
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AVEKILL A. LIEBOW. M.D.. Chair- ntan, Depar,nunt of Pathology. Yale University School of Medicine, New Haven. CT. IRVIN E. I.IL'NEK. Pu.D., Professor oJ BiocAtruiury, University of Mtnnesota, St. Paul. ESTEN O. LINDSETII, M.D.. PK.D.. St. Jouph's Llospital Research Laboratory. St. Paul. MN. ROBERT H. LINNELL Pe.D.. Associ- are Pru/usur a/ CAenrissry. University of Vermont. 8urhagtoo. HERBERT L. LOMBARD. M.D.. M.P.H., A(Jiliote, Cancer Rrsearclr h,- stuwr. New England Deaconess tius- pital, 8oston. J. P. LONG. P(r.D., Professor of Phar- n,acoluYy, State University of lowa College of Medicine, Jowa City. CLAYTON G. LOOSLI. PK.D., M.D.. Nastinps Professor of Medicine and Pathology. University of Southern Cali- Lornia School of Mcdicinc, Los Angclcs. DONALD B. LOURIA. M.D.. Assoclate Professor o/ Medicine, Cornell Uaiver- rity Medical College. New York. KENNETH MERRILL LYNCH. M.D.. Se.D.. LLD. Pro/rasor Emeruus of Pathology and CAancrllor, Medical College of South Caroliaa, Cbarlcstoa INES MANDL. Pu.D. Assisranr Pro/u- soe of Biochurris,ry, Columbia Univcr- sity Collc`e of Pbysiciuu 1t Surjeons, New York. JOHN H. MANHOLD. J.., D.M.D.. Profesbw and Director. Departnrenr of Pa,bolu y arrd Oral Diasnosis, New Jersey ~ollcSe of Medicine and Den- tisrry, Jersey City. DAVID E. MANN. Prr.D., Associate Professor o/ Phainracolop, Temple Univusity Scbool of Pharmacy. Phrlr dclplua. FRANK ARTfiUR MANNING, M.D.. Asristanr Profrssor o/ Obs,etrin and Gyneculofy, Woroeri a 1/osprtal, Los An4eks County/Uaveruty of Suuthern C.Lfornia Medical Ceata, Los AnAcks. JOHN P. MANOS. M.D.. InstruCtor in Yirolury and Bacrniolory, Medical College of South Carolina. CDarks(on. CLIKIS'fOPLt1:K M. MAR'1'IN, M.D.. Is..ntunt Pr../a'suur ./ AJrd,une and Doc.'l.N, l)n-,un u/ In/e.'t1UYi Dis- rasrs, Sctun Ilall College of Medicine. Jcncy City. R. RUSSELI. MAKTIN- M.D., ProJeuar o/ Mrdicute, and Aftrrubiulugy .4 Fiu- n,unuloLy, Baytur College uf Mcdicinc, Ilouston. REGINALD G. MASON, L.1.U., 1'u.D., Proles.ur and Cha,nnun, Depuquun, o/ Podwloyy. Univcr.ity of Utah Cul- Icse of Medicine. Salt Lake City. MASON RESEARCH 1NS"LITUTE, Wurccucr, MA. DONAI 1) 1. MASSARO. M.D.. Assuci- .ue 1'ru/usur of Alydicine, George Washington University School of Mcdicinc, WashinAton, DC. CHARLES McAKT11UK, Ihr.D.. Psy- eholugiss, University )lealtJ, Serviau, Ilarvard Univcrsity, Cambridge. MA. CHARLES B. McCANTS, Prr.D., Asso- ciatr Professor of Suils, North Caro- lina State College School of Atiricul- turc, Ralci&h. GERALD E. McCLEARN. Ptt.D., Di- rector, lnstitute Jor Behavioral Ge- netics, Dr rrnent u/ PsycbuluRy. Uni- vcrsity ofv Culurado School of Phar- macy, Boulder. HENRY C. Mt'GILL., )a., M.D.. Ac,int; Ilrad, Dr•purrn,rnt of PwGuluyy, l.uuir siana State University Schrx:' of McJi- cine, New Orleans. HENRY D. McINTOSl1, M.D.. Pro/es- sor o/ Medicine and Director. Cardio- vascsolar Laboratory. Duke University Medical Center. Durham. NC. FORDE A. MeIVER. M.D.. Associate Profes.or u/ PatAuloYy, Medical Col- Iete of South Carolina. Charleston. EDWARD McKEE. M.D.. Professor and Acting C)ra,rruan, Depurmtent o/ Pathology. Medical College of South Carolina, Charlestun. KIiL.LY T. MrKL•1:, M.D.. Asswwrr Profeuw uf A/edu•lne, Medical Cul- kse of Suuth Carulina, Cbarlestun. IIERBF.KT Mr'KF.NNIS- /K., Iht.D.. Rr- srarch P/U/rJNN of PYIAVLw,v. Llnrvcr- sily of Miaou School of Atedlcine, Mwmi, FL. 112 VL(TOR A. McKUSICK, M.D., Pro/cs- sur o/ Mrdicine, 7be Johns Hopkins Univcrsity School of Medicine. Balti- morc. ROSS L.. McLEAN, M.D.. Associate Pro/rsiur of Afrd,cine, Emory Univcr- sity School of Medicine. Atlanta. WILLIAM F. McNARY, Jst., PN.D.. As- suciate Pru/ensor of Anatomy. Boston Univcrsity School of Mcdicinc, Boston. Nl?AL L. McNIVEN, Psr.D., The Wor- ccstcr Foundation for Eapuimcntal Biolojy,Shcwsbury, MA. HANS M1411; .K, D.V.M., Srnior StaQ Sri- enrur, The Jaelsun Laboratory, Uar I /arbor, ME. JULIA MEYER. Pw.D., Assoriatr Pro- lessor of Oral Parbolorr, University of Illinois College of Dentistry. Chicago. DOV MICHM?L1, Ptr.D., Assiuaru Pro- /rssor u/ BiucJ,en,istry and Surrrry, University of California School of Medicine. San Francisco. MICROBIOLOGICAL ASSOCIATES. INC., 8c,hesda. MD. BERNARD J. MILLER. M.D.. Assisrant Professor of Anatomy. Jef(erson Medi- cal Collcge, Philadelphia. JAMES G. MILLER, M.D.. Pn.D., Pro- /rssur of Psychiatry and Psychology; Direcu.r. A/enral J/ealrA Research In- surure- University of Michigan. Ann Arbor. JERALD A. MITCHEL.1.. Pu.D.. Assu- riarr ProJeasw of Anatuury, Wayne State Univcnhy School of Medicine. Dctroit. CHARI.I:S MIITMAN. M.D., Director. Departnunt u/ Respiratory Diuaser, ('ity of flupe National Medieal Cen- acr, Duarlc. ('A. Hll(111 MON'IGOMfKY, M.D., Asso- .wu Pr../nsur u/ Alydr.ine. Univenity of 1'cun.t'Ivan,a School of McdiCsnc. Phdadclp/na. 1'. (I'It AIONYIa)AlVKl'. )a.. M D. Pr..Jnvw u/ )Su/udut;y. Ilnivcnity of Tesas Suullrwcatern Medical Sehoul, Ua/t... GIiOKGIi li. MlK)KL?, 161 D., Ih,.U. D,- rr.l..r. Ku.wcll P+rl hle+uurial Insti- tntc, 1lutfalu. NY. 113 KENNIiTH M. MOSER. M.D.. Assistant Pro/esror uf Medicine, Georsctuwn University Medical School. Washirrg- ton, DC. f/URLFY LEE MOTLEY, M.D., Profrs- sor of Medic'.ne and Direcror, Cardro- Resp,ratory Laboratory. University of Southern California School of Mcdi- cinc, Los Anscks. EDMOND ANTHONY MURPHY. M.D., SC.D., Associate Pro/essor of BiastalisNCS and Mrdicine, The )ohna Hopkins University School of Mcdi- cinc, Baltimore. WILLIAM S. MURRAY. Sc.D.. Senior Staff Scientist, The Jaekson Labora- tory, Bar Harbor, ME. RICHARD L. NAEYE, M.D.. Professor and CAairn,an- Deparrn~enr of Pathol- osy, Pcnnsylvania State University Col- Iep of Medieine, Hershey. GEORG B. NEURATII, PH.D.. Micro- analytical [.abora,ory, fiamburt, West Germany. ALBERT H. NIDEN. M D. Professor of Mediune- Drew Pustxraduate Medical School and Univenny of Southern California; Clnel. Puln.vnary Diseau Secnon, Martin Luther King Hospital. Los Angeles. OAK RIDGE NATIONAL LABORA- TORY. Oak Ridge. TN. DONAL D M. PACE. Prr.D.. Professor of PAyu..loyy and Dlrec,or, Insntatr /or Cellular Research. University of Nebraska. Lincoln. BEVERLY PAIGF:N, PuD. Cancrr Re- uwrb Srirruiat 1', Kuswcll Park Me- morial lastitute, BuQalu. KFNNFTLL PAIGEN. Iht.D, Dirrrrw, Drp,nn,rnt u/ Al,dr'an6.r Biul.rY)'. Hcal,h Rcae:.r.h. In.., Kuswell Parl Divi.ion, BuA:rlu. ALBERT B. PALMER. Prr D.. Assistant PruJrssur u/ Patb.,loyy. Universiry of Toledu. 7 ukdu- ( )l 1. ROSL' MARIf: PAN(:ItOKN. A1 S.. As- su,an, Paal Tr.l.n.d.ynr and Lrcrnrer, Dr~wr,rnrnr ../ F u.al .11 Ifrlrr and 71'I'A- n../..w. Univcrsrty of C.hlurn,a, D.rvis. JOIIN W. I'ARKI1K- M.D. Arsociate Pro/i INN ../ Pwh,dugy, Umvcrsity of S.wthcrn ('alifurnr. S.boul of Mcdi- :uk. Lus AnScles. T C ~ T i
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a MARY STEARNS PARSHLEY, Pu.D., Assismnr Pru/sasur o/ Anaronry in OA- srarica and Gyntcoloiy, Columbia Univcrsity Colk`e of Pbysiciana k Sur- aconz. Ncw York. EDWARD W. PELIKAN, M.D.. Chair- rrran, Deparrmenr of Pharmacoloty and EAptrintrnlal TAerapeurics, Boston Univcrsity School of Mcdicine, Bostoa. CARL W. PIERCE, M.D., P((.D., PruJes- sw o/ Pathology und Alicrubluluyy-lrn- rnunoloJy, Washin;lon University School of Medicine; Director of Puth- olosy and Laboratory Medlcine, The Jewish Hospilal of Sr. Louis, St. Louis. MALCOLM C. PIKE. Prr.D., Professur of Cunununiry Medicine and PrJsnr- riu. Univcrsity of Soulhern California School of Medicine. Los AnSclcs• OTAKAR J. POI.LAK, M.D.. Prt l)., l:'stcuGvt Duectur, Dovcr Medical Kc- scarch Ccn(cr, Inc.. Dover. DE. MORRIS POL.LARD, Pu.D., Direcror. Lohwnd Laboratory. University of Nwrc Damc, Noirc Dame, IN. C. M. POMERAT, Prr.D., Director of Biolutical Restwch, Pasadcna Founda- tion tor Medical Research, Pasadcna, CA. S. N. PRADHAN, M.D.. Prr.D., Prufts- sw u/ PJwrnwcology, Howard Univer- sity Collcsc uf Medicinc, Wasbinjton, D.C. H. R. PRATT-THOMAS, M.D. PruJrs- sor of Pathology and Dean. Medical College of South Cuolina, Charlcuon. PROCESS Jr INSTRUMENT CORPO- RATION. Brooklyn. NY. MARTIN S. PROTZEL, D.D.S.. Chief, Depurrnrenr of Oral Patholory, Ncwuk City Hospital- Newark. NJ. WALTER REDISCII. M.D.. Associatr Professor of Clinical Mrdicine, New York University School of Medicine, and NYU Research Service, OolJwatcr Memurial Ilospiul, New York. TIMOTHY J. REGAN. M.D., Pru/ruw u/ Aledicinr; Director. Divisiun u/ CurJiurasa'alar Diseaus, Collcge of MeJtcinc and Dentislry of New lcrxy, New Jersey Mcdical School. Ncwark. W1LI IAA1 KE(iF:1.SON. M.D.. Professor and Chairtuun, !)rparrntent of A1rJ,caf Onculupy, Mcdical College of Vir`inia, Riclunurrd. LYNNE M. REID. M.D.. fYulbarh Yru- /essur of P,uhulu,;y, IlarvarJ A1cJical School. Busbm; Chair,nun, Departntrnr of Pathulu.y. ChilJrcn's Hospital Medical Centcr, Bouon. HOBART A. KEIMANN, M.D.. Pru/es- w,r of Mrdicirne. Habnernann Mcdical College and Iluspital, Philadelphia. KOLI.AND C. RF.YNOLDS, M.D., As- sutunl Pr.,/ruw u/ Pathulogy. Univer- sitY uf 1'ecas Suuthwestcrn hlcdical Schuul, Dallas. VICTOK KICIIAKDS. At D., Chit/ uf .1urgrry, I',c.bylcrian Medical Ccntcr, San l-tan.:ixu. ARNIS KIC111'IiKS, 1'u.D., A„unwr Pru/nwr ul PathulaAy, l)nrveraty of Southcr n Caltfornia School of Medicine, Los Ansclcs. WILLIS 11. Kll?SI'.N, 1'nD., S'rniur Bio- chenwl, l.tfe Scintct, Division, lIT KCKarlh Insutnnc, ('bicagu, DANllil. B. K1F'KIN. Iht.D., Aui,t,uu Yra./rs,ur ../ C'hcnu.,d Bw1uYy. Tbe Koclcfcller Unrvcrsity, New Yurk. K. il. KIGDON, M.D. Professor uf Pa- tlwlugy. Univcrsity of Teaas Medical Branch, Galveslun. SYDNEY C. K1TIliNBI_KG• Pu.D., Professor u/ Hacttriulory. Univctsily of Southern California. Los Angeles. BENSON B. KOt, 161 D., Aasuciate Pro- /c»ur u/ Surrery; Ch,cl. Cardiac Sur- srry, Univcrstty of California School of Medicine. San Franciscu. JOSI=PH II. KUGEKS, hf.D.. Holy Name uf lcws 11u.prtal, Gadadcn- AL. KOBIiKT C. ROSAN. M.D, Assuciare Pru/rssur of PuthulLry and PrJ,utrics. St. Louis University S.hrwl of Medi- cine; Aa,u.,ute Pu)hulufist, Ca(Jinal Glcnnun Atemwial Iluspqtal for Chd- dren, St. Louis. CHARLLiS L. ROSE. PH.D., Clinic Uurc. rw; Directur, Nurmaurr Aginl S)uJy, Veterans AJnanistranun Dutpatrcnt Clinrc, IJustun. JOHN A. KOSL•CKANS, 1'u.U., Asu- c,arr Professor „/ Pharrnoc., McJt- cal Collcge of Virginia. Virginia Com- nronwcaltb Univcraity, Richmond. JOHN K. ROWLANDS. PN.D.. StaQ Scitntisr, Southwest Rcsearch lnatitutc, San Antuuiu,'I*X. BENJAMIN A. KUBIN, PN.D., Auistant Pru/essur uJ Public lltuld,, Baylor University College of Medicine, Huu- stun. KONALD P. KUBIN. Prt.D.. Professor of Phannaculury. Medical College of Virlima. KichmunJ. alf•NKl' I. KUSSI?K. M.D.• Prnidrnr, 'I he Kus.el Foundation. Inc., Statcn 1s1auJ. NY. W. ('. KUSSIi1I., M.1).• llnivrtsity of 'Icaas McJ,cal Ccsucr, Iluuslun. WAYNP 1. RYAN. Pu.U., Pru/rr,ur of It,,,ahrv,u.tuy. Univcrntty of Ncbraska Cullegc of McJionc- Omaha. 1'1• 1 NK V. SALISIIUKY. M.D., Prs U., /JtaJ, Iwrrnn•t Trtuuntnt Center. Samt Juxph I lu.pital, Hurhank, CA. PAl/l. SAI l MAN. Pu U.- A,si,ru,u Pro- /ruw u/ Bw.htn,isrry and Nutritiwr. lJnivcrsity of Southern California School of Medicine. Lus Angeles. UI KICIJ 11 N('l1AEPPI, M.D., D.rec- tur of Nrar.,phurnraculuCy. Mason Rc- scarch Inslmuc. Wutccster, MA. JOK(il!N U. SCI/L1:(iLL. At U., Pu.D.. Prul"s,ur nn,l Chuonrun. Drpartamenr u/ S,uArry, Tulane University School of MeJtune, New Urlcans. Al VIN I(. S('IIMIDf, Pu.D.. Drrtctw „/ (',•un.rl,ng, Tults Univunuy, Mcd- I.uJ. hIA. JAKOB SCHhtIDf, M.D.. Pu.D.• As- srstuat Yru/es,w uf 14wLnm.ury. D,- rn,un u/ U,uluq:rcol Sc,taars, S(a1c l/uivctsny of New YurL at Stuny uruuk, Stuny Bruuk. I.IAA(' St'l1(UK, 1) U 5. 1'rr I) • U Sr'.. llcm,. Umvcr.rn ut Illtmns l'ullctc of Ucnu,uy, ('hr:agu. Sl'K11'I'S r'I.INII' ANI) KI[1IiAK('11 IOUNUA I I(1N. 1 a lulla• ('A. MAUKICE S. SECJAL, M D.. Clinical ~ Pru/r.vur uf A1tJic-inr, 'lufts Univcr- ~ sity Schoul of Medicine; Dirrcror, Dc- C41 parttnrnt u/ /nhalariun Thtrupy, Bos- tun City llospital, Bastnn. T"1 CARL C. SL'LTZEK, Prr.D. Honorary Ruearch A,sociatt, Peabody Muscum, Harvard University, Cambridge. MA. LUCIO SEVEKI- M.D.. Dirrcror and Dran, lnstitutt of Anatu.rry and Pa)hol- u y, Division of Cancer Rcscuch, 1 ~ ~ U lfnivcrsily of Pcrugia. Perugia. Italy. ® CIIAI(LES R. SHAW, A1D.. Chief. Sec- riun u/ AlrJicul Genrtics, M. D. An- derwn Huspital and Tumor Institute; Pro/rs,w ul SruloYy. Thc Univcrsity of Tesaa at Houston. Ilouston. CIiAK1 IiS I'. S11NKWOnD, M D.- A,- si,ranr Pru/ruur u/ Radiolury. Urtiver- sily u( Kuihcsrcr Schuul of Medicine anJ Ucnustsy, K..chcsacr, NY. SI1O1I SIIIBA'fA, M U.• Pu.D. Pro/ts- rur „/ Phurnruralugy, Univcrsity of Ilawati School of McJrcinc, Honolulu. UAVID L. SIMON. M.D • lnsttuct.n tn lnrrrnal AJedacu,r, Cincinnati General Huspilal, Cmciunau. FKIK SKINHQJ, h1.D.• Chief. Depurr- tntat of NturoluYy. Bnpcbjcrg Huspi- ul, CopenhaAcn. NA'1H.1N H. SLOANIi. Iht.D. Ya.Jn- wr uf B,nchr,nutry. The Univeraaty of 7'cnnc»ce ('entcr fur the Hcaltb Scicnccs, htcmpbrs. l HI'OWKI. A. SLOI KIN, Pu D.. As- sissunt Professor of PhwmaculuYy. Dukc Univcruty Mcdical Ccntcr, Dur- ham. NC. GEOKGF: W. SMF7TERS. M D., Arso- ciate in P.uhuluYy, Nwlhwestcrn Uni- vcrsity Medical School. Chica;o• Gl:NF Itl. SMITII. I'rr.D • Arnivunr Pru- /r,wr u/ 1',yrh,.lugy, ilarvar.l Alediaal School. Maasachuxtts Gcncral Huspi Ilusluo. lal. L.UCIt Ii SMI LII• Pu D. P.n/r,rw n/ IlHahrnu.uy. U.ttm.w/h Medical Y.h..ul, N11 LOlI1S A Sl)1(11•1. All). /I/an,he 1'. Ln•r D,urnj;unhr./ Srrvnr PIU/rss.M, ../ AlrJn.nr. Xr stun b l.,l'iJ f,IAJIatUry. I l•nlpk l)n,- ver.ny Ilcalth Sacn.cs Ccntar, Plula- Jclpbta. 114 115
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1 v 2 SHELDON C. SOMMERS. M.D., Doec- tur uJ Lab.xaturits. Lerroa Hill Hos- pital; Clinical Proltssor u/ Parhology, Columbia University College of Physi- cians i Surgeons. New York. ERNEST SONDHEIMER, Psr.D., Ano- ciarr Professor oJ Biochemurry, Col- kgc of Forestry. State Uoiveraity of New York. Syracuse. T. M. SONNI:BORN, Prs.D., Distin- JNd StrYlct Professor o/ Zoology. Mana Univcrsity, Bloomington. SAM SOROF, Prr.D., Head. DrPartmtnr ol Macromu/ecrlu CAtm4try, Thtt lnuituta for Cancer RsacarcJ4 PiRa- delpaia. SOUTHWEST RESEARCH INSTI- TX i TUTE, San Anton o. . IRENE Y. WANG. Prr.D., Assistant Pro- Jtssor o/ Basic and Clinical lmrnunol- oly and M,crobioloty, Medical Uni- vcrsity of Soutb Carolina. Chulcuon. E. D. WARNER. M.D.. Pro/tssor o/ Pa- rholusy, State University of Iowa Col- lcgc of Mcdianc, lowa City. Si1lEl.DS WARREN. M.D.. Director of Laburatur,ts. C'anctr Research Jn.rri- rurt, New England Dcaconcsa Ho.p- ral, Bos+on. YASUSHI WATANABE. Prr.D.. Aiso- ciate Mnnbrr, The Wistar Instituta of Anatomy and Biolo4Y, Philaddpbta. BARBARA K. WATSON. Pe.D. Ass(sr- ant Bacttriulotist. Massachusctta G+n- eral Hospital; Research Assocbtr in DUANE G. WENZEL, Psr D. Professor and Chairman, Drpartmtnr of PJwrma- culuyy and Tusicolujy. The University of kanaaa Scbool of Pharmacy. Law- rcnu. THOMAS C. WESTFALL, Ptr.D., Pro- lessor a/ PharmaculuYy. University o( Virginia School of McdKlnt, Chat- lottcavilk. FREDERICK E. WI(ISKIN, M.D. C.M.. Director. Diruion o( 1lralrh and Ptr- sonahty EQrihbriun,, The Aga Center of New Ea{lanQ Inc.. Boston. JAMES A. Wi1.L, D.V.M.. Ptr.D., Pro- /tssar and CAa4man. DtPartmtnr of Y`a`d1q kladiso~ • University of wik co ew o . Bucruiolusy and lmmunololy, Har- t B h l ROGER 1. WILLIAMS, M.D.. Professor DAVID hl. SPAIN. M.D., Director. Dr- I.IE SHA TSAI, Pu.D., Rtsturrh A,ro- os on. oo . vard Medical Sc of Chemistry; Director. Clayton Foun- pwuaeru oJ Puthulusy. The Brookdale ciate in Pathology. Yale University LEE W. WATTENBERO, M.D., Pro/rs- darion Biochemical Institute. Tbc Uni- i f T Hospital Center. Bruoklyn, NY. School of Medicinc, New Havcn, Cf. sor o/ Parholosy. University of Min- li Mi S h l n. vcrsity o caas. Aust ALI:XANDER SPOCK. M.D.. A»i,tanr GERALD M. TURINO. M.D., Pru/t„ur s. nocapo ncsota Medical c oo . DANIEL H. WISEMAN. h1.D.. Assur- i Pru/t,sor u/ Ptdiatrics, Duke Univer- siry MeJical Center. Durbam, NC. oJ Medicine. Columbia University Cul- lege of Physicians 4 Surgcons, New JOHN S. WAUG/(, Pu.D.. Pro/tssor oJ Chtmi,rry, Massachusetu InuituWa of ant Pro/essw of Ptdiaures- Univers ty of Southern California; Children i Di- l C G l York. Cambridge. Technology es viaion, Loa Ange ounty enera FREDERICK J. STARE. M.D.. Pro/rs- . Hospital. Los Anacks. sur u/ Nurririun, Harvard University D. M. TUKNFR, 1'u.D, fltuJ, Dtpart- KICHAKD (.. WECIiS1.ER, M.D., Chn- School of Public Health. Bustun. n,tnr u/ Drug A(Nabulistn and Bio- ical Physiulotur, Monte6we Huspital GEORGE WOLF. D. Psus., Pro/tssur o1 chnni.stry, ((atlelun Lalwraturica L'u- Pdubwab. lmututc of Rcscarch Physiological Chemistry. Department C. I/AKOLD STEFFEF., M.D., Dirtctor ropc, Ltd., llattubate, Yorkshire. L•np , Massa- of Nutrition and Food Science oJ Labuiututits• Methodist Hospital. land. JOIIN V. W(:11., M.D.. Assl,rant Pro- . Cam- cbuscrts Institute of Tcchnoloajy A(cmphis. Jrs.wr o/ MeJinnt. University of Colo- . bridse. Assuciore STRONG M JACK P D UNION CARBIDE COKPOKKf7ON, radu Medical Center. Deaver. . . . ., Pru/tauw uJ Parhulucy Louisiana State Nuclear Divisiun, Oak KiJac, TN. 1. EDWIN WOOD. M.D.. In,rructor in , Unrversity Schoul of Medicine. "New UNIVERSI7Y OF SAN FRANCISCO, A. WEINSTOCK. PtsD., Research Biu- Life Scitncts Division, IIT chuaut MtJit,nr, Boston University School of Boston Medicine Orleans. San Francisco. . Research Institute. Chicago. . . MARION B SULZBERGER Pro- M D Assistant SUMNER WOOD M D Ja , . . .. Deparrmtnr ../ /rssor and Chuirnwn UNIVFkIITY OF SOUl71F.KN CALI- RUSSELI. W. WELLER. M.D.. Pathol- . .. . .. Professor a/ Patholo[y The lohm , New Dtrnratolojy and Syphilology 1•()KNIA, Los Angeles. uYur, Memorial Hospital of Chcater . Hopkins University Scbool of Medi- . York University-Bcllevtre Medical Cen- Pru/ts,urand ELLIOTS. VI:SELI. M.D. County, West Chester. PA. cine, Baltimore. ter, New York. , , Chairman Dt/wrtmtnt of Phunnacul- A. STANLEY WELTMAN, Prr.D., Asso ialt KENATO TAGIUKI As o Pu D , uYy. Pcnnsylvania State University Col- ciart Pru/tssur oJ Pharmacology and JOHN P. WYATT. M.D.. Pru/t,sor o/ , . . . r d P P h l G t Icsc of Medicine. Milton S. Ilcrshcy Re,tarch. Brooklyn Colleje of Pbu- Parholory, Saint Louis Univcrsiry ru/essur o/ ua syc o ogy. ra e School of Busineu Adminiuration, Medical Center. Hcrshcy. macy, Brooklyn. NY. School o( Medicine, St. L.ows. Hxvard University. Boston. DAVID W. TALMAGE. M.D., Dirtca.r, Wrbb-Wuring Lung /nsritutt, University of Cdorado Medical Center. Dcnvcr. LYNN M. TAUSSIG. M.D.. Asuciutr Professor and Aswciatt Chairn,an, Dtpwtmtnt ../ PtJiasrics, Arizona Health Scicaces Ccntu, Tucson. MARC D. THAMES, M.D.. Senior Rt- search Fellow. Mayo Clinie and Foun- datiun. Rocbcucr, MN. BRANISLAV VIDIC. D S, 1'rn/t~au ..J Anubnny. Georgetown llnivcnuy Schools of Atcdiunc and Dcntistry, WashinStoa, D.C. KOAIF<) A. VII)l)NE- M.D.. Auuuure Piu/es,ur u/ l'urhutuAy. Yale tlniver- siry School of Medranc. New Ilavcn, CT. PFfEfr K. V(X:1. Pu.D.. Pruftnwr u/ Ali.rubiulut;y, llnrvcn,ty uf Wash,ns- tun Schoul uf Med,cine, Scattlc. SIMON If. WENDFK, Pu.D., Research Pru/t,sur o/ Bowhtr.u,rry, Univcraity of Oklahoma. Nwman. KOII YOSHINAGA. PnD.. Center for Population Research. NICHD, National I nstitutes of Heallh- Bethcsda, M D. CAROLINI: 111-Dk:LL TI IOMAS, M.D.. Pru/rs,ur f:'nurilus u/ AttJicint, The Juhrts Hupkins Univcrsity School of Mcdicinc• Baltimore. JEROME F. T1IOMAS, Prr.D., Pru/t»ur n/ Sanitary £nrineerinr, University of California. Bcrkeley. JAMES F.. P. TOMAN, Pu.D.. Pro/ts- sur and Chairman. Dtpornntnr oJ Phar- u,acololy, Chicago Medical School, In- atilutc (or Medical Research. Chicago. ANDREW M. TOME.TSKO, P,t.D., Pres- ldenf and Director u/ Research, Liiron Laboratorics, Ltd.. Rochcster, NY. JANET TRAVELL. M.D.. Associatt Professor u/ Clinical Pharmacology. Cornell University Medical College. rk N Y ® . Ilb 117
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.i . INDEX OF SENIOR AUTIIORS Abranu. W. R., 32 Albanu, W. A.. 22 AnJersson, K.. 61 Antoniues. H. N.. 51 Arnaout, M. A., 56 Barrincau, L. L.. 24 Beatty. K.,'_9. 32 Ucrnucin. L. R., 52 Uhaltacharya. A., 84 Braughlcr. J. M.. 65 Brown, J. K., 33 Busbec. D. L., 66 Bush, G. A.. 76 Carp, H.. 28 Chakrabarti, S.. 7 ChaturveJi, A. K.. 73 Cualson. D. W.. 16 Coughlin. S. R., 49 Dancs, B. S.. 20 Davies. B. D.. 62 Diala, E. S.. 12. 14, IS Duwney, H. F., 55 Ellis. J.. 63. 64 Erickson. C. K.. 66 Fost%:r. 1. A.. 23. 25. 67 Fox. R. U., 36 FricJman, G. D.. 89. 90 Galanakis. D. K., 35 Gcukas, M. C.. 48 Giclcn. J. E.. I 1 Harnmcr, R. E.. 70 Harada, R. N.. 40 Hartwig, J. H., 43 llcgarty, K. M.. 56 Ho. M-K.. 86. 87 Huliman, R. M., 14. 15 Holmberg, L. A., 85 Hwang. K. K.. 70 Jaiswat, R. K.. 74 JanoR, A.. 30 Kaprio. J.. 91. 93, 94 Karr, S. K.. 67 Kcith, 1. M., 47, 48 Koskcnvuo, M., 92 Kouri, R. E.. 10 Kremers, P.. 12 Kucttrscr, K. E., 17, 18, 19 Lanlinvainio, H.. 90.72 laryman. C., 27 I.arwn, R. A., 59 I.awrence. I?. C.. 8o IAC. W. M. F.. 81 Lca~c, C. C., 52 Li. H. C.. 69 Lu. C. Y.. 87. 88 Lynch. H. T., 21 Machcr. U. A.. 81 Marks, M. J.. 61 Marshall. M. V., 8 Martin, B. R., 60 Maruyama. K.. 43 Mathcwn, N. R.. 31, 77 Otsch, F.. 72 Owcn, A. l. 111, 50 Pagini, F. D.. 35 PaiLcn, 8., 10. 68 Partincn, M., 94 Pauli. B. U., 19,20 Platt. K. L., 71. 72 Powcrs, J. C.. 26, 27 Kankin. J. A.. 82 Rasp, F. L.. 36. 38 KcrWi. A. H.. 17 Kcilly, C. F., 31 Repinc. J. E.. 37. 38. 39 Rhim. J. S.. 23 Kubnitz. 1. E.. 13 Ryan. J. W., 57 Kyan, U. S.. 58 S+stry, U. V. K., 75 Schapira. M.. 54, 55 Schuclkc. G. S., 22 Sershen. 11.. 62 Shasby, D. M.. 39, 41, 42 Snodgrass. D. K., 9 Snyder, D. S.. 88 Springer, T. A., 83. 84 Stosscl, T. P.. 45. 46 Tatc, K. M.. 41 Taussig. L. M., 47 Taveira D. Silva. A. M.. 34 Thompwn. J. A., 73 Towcll, J. F.. 63, 67 Travis, J., 29 Valcriuc, N. H.. 44 Van C'antturt, J., 8 Virca, G. D.. 78 Wharton, J.. 82 White, R.. 25 Wiggin., R. C., 78 Williams, 1.. T.. 53 Yachnin, S., 59 YancrJa. H.. 33 Yansamwo. T., 79 Yin. H. A.. 44 Yu.hinrur.r, T.. 27 118 INDEX OF PRINCIPAL INYFSTIGATOR3 Accru, M. U.. 60 AntuwaJcs, 11. N., 49, 50. 51. 52. 53 ttiswa., D. K.. 7 Braubhlcr, J. M.. 65 Uusbce. D. L.. 8, 9. 66 Cuchranc, C. G., 78,79 Cullins, A. C.. 61 Colman, K. W., 54, 55 f)uwney, li. F., 55 Erick.on, C. K.. 66 Erwin, V. G., 63, 67 Fostcr, J. A.. 23. 24. 25, 67 FrieJntan, (7. D., 89,'!0 Fuxe, K.. 61 Geuka., M. C., 27, 48 Cicasncr, T.. 68, 69 C:iclcn. J. 1:., 8, 11, 12 Gold. W. M., 33 Hanrosh, P.. 34. 35 Henry. C. J., 70 Ilolfman, K. M., 12, 13. 14, 15, 16 I lain•rcki, 1. 1.., 56 / luss, W.. 61. 63. 6-0 Janulf, A., 25. 2x, 30, 35 Kcith, 1. M.. 48 Kouri, K. /?., 10 Kuctlncr, K. 1:., 17, 18, 19, 20 Lajtha, A., 62 Lrwrcnce. E. C.. 80 Lionctti, F. 1., 56 Lynch,H.T.,20,21,22 Macher. 8. A., 81 Meicr, H.. 23 Mitchell. J. A., 70 Ocsch. F., 71, 72 Paigcn, K.. 10 Petersen, O. R.. 73 Polak, J. M., 82 Kantasalo, 1., 90, 91, 92, 93, 94 Kcpine, J. L'., 36. 37. 38, 39, 40, 41, 42 Reynolds. H. Y.. 82 Ryan. U. S.. 57. 58 Sastry, B. V. R., 73, 74. 75 Springer. T. A.. 83, 84. 85, 86, 87 Stoascl, T. P., 43. 44, 45. 46 Tau»ig, L. M.. 47 Travis, J., 26.27.29. 31, 32, 76, 77, 78 Unanrrc, L. R.. 87, 88 Wcinbaunr, G., 32. 33 Will, 1. A.. 47, 48 Yachnin. S., 59 119 ®

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